U.S. patent number 7,429,596 [Application Number 10/871,732] was granted by the patent office on 2008-09-30 for 1h-pyrrolo [2,3-d] pyrimidine derivatives and methods of use thereof.
This patent grant is currently assigned to The Regents of the University of California. Invention is credited to Raynard L. Bateman, Alma L. Burlingame, Stephen G. DiMagno, Kirk Hansen, Kevan M. Shokat, Masahiro Tanaka, Chao Zhang.
United States Patent |
7,429,596 |
Tanaka , et al. |
September 30, 2008 |
1H-pyrrolo [2,3-D] pyrimidine derivatives and methods of use
thereof
Abstract
This invention generally relates to pyrazolo pyrimidine
derivatives useful as inhibitors of short chain
dehydrogenase/reductase (SDR) family of NAD(P)(H) dependent
oxido-reductases. The invention further relates to pharmaceutical
compositions and methods of preventing or treating disease with
1H-Pyrrolo[2.3-d]pyrimidine derivatives. More specifically, the
invention relates to a 1H-Pyrrolo[2.3-d]pyrimidine which is a
compound of Formula I or II: ##STR00001## or a
pharmaceutically-acceptable salt or prodrug thereof; wherein: Y is
N or CR.sub.5; Z is NR.sub.3R.sub.4, halo, H, OH, alkyl, alkyloxy,
or haloalkyl; and R.sub.1a is indolyl, thiazolyl, benzyl,
biphenylyl, thiophenyl, pyrrolyl, or phenyl, wherein said phenyl is
substituted with at least one of OH, --NR.sub.3R.sub.4,
--C(.dbd.O)NR.sub.6R.sub.7, --CN, NO.sub.2--C(.dbd.O)OH,
--C(.dbd.O)O-alkyl, (C.sub.1-C.sub.4)alkyl, halo, haloalkyl or
haloaryl; and wherein said indolyl, thiazolyl, benzyl, biphenylyl,
thiophenyl, or pyrrolyl is optionally substituted with OH,
--NR.sub.3R.sub.4, --C(.dbd.O)NR.sub.6R.sub.7, --CN, NO.sub.2,
--C(.dbd.O)O--R.sub.3, (C.sub.1-C.sub.4)alkyl, halo, haloalkyl or
haloaryl.
Inventors: |
Tanaka; Masahiro (San
Francisco, CA), Zhang; Chao (San Francisco, CA), Shokat;
Kevan M. (San Francisco, CA), Burlingame; Alma L.
(Sausalito, CA), Hansen; Kirk (San Mateo, CA), Bateman;
Raynard L. (San Francisco, CA), DiMagno; Stephen G.
(Lincoln, NE) |
Assignee: |
The Regents of the University of
California (Oakland, CA)
|
Family
ID: |
34526201 |
Appl.
No.: |
10/871,732 |
Filed: |
June 18, 2004 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050085472 A1 |
Apr 21, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60480501 |
Jun 20, 2003 |
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Current U.S.
Class: |
514/265.1;
514/252.16; 544/117; 544/280; 544/244; 514/81; 514/234.5 |
Current CPC
Class: |
A61P
35/00 (20180101); A61P 3/04 (20180101); A61P
35/04 (20180101); C07D 487/04 (20130101); A61P
29/00 (20180101); A61P 3/00 (20180101); A61P
35/02 (20180101); A61P 3/10 (20180101) |
Current International
Class: |
C07D
487/04 (20060101); A61K 31/497 (20060101); A61K
31/519 (20060101); A61K 31/5355 (20060101); A61K
31/662 (20060101); A61K 31/675 (20060101); C07D
413/06 (20060101); C07F 9/38 (20060101); A61P
35/02 (20060101); A61P 35/04 (20060101) |
Field of
Search: |
;544/280 ;514/265.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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812366 |
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Apr 1959 |
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GB |
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WO 93/22443 |
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Nov 1993 |
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WO |
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WO 97/28161 |
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Aug 1997 |
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WO |
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WO 98/41525 |
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Sep 1998 |
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WO |
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WO 98/41525 |
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Sep 1998 |
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WO |
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WO 00/17202 |
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Mar 2000 |
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WO |
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01/19829 |
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Mar 2001 |
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WO |
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03/020880 |
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Mar 2003 |
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WO |
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WO 2005/097800 |
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Oct 2005 |
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WO |
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|
Primary Examiner: Coleman; Brenda
Assistant Examiner: Moore; Susanna
Attorney, Agent or Firm: Townsend and Townsend and Crew
Government Interests
This invention was made with Government support by Grant Nos.
AI44009 and NCRR RR01614 awarded by the National Institutes of
Health. The Government has certain rights in this invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority to U.S. Application No.
60/480,501, filed Jun. 20, 2003, the entire disclosure of which is
incorporated herein by reference.
Claims
What is claimed is :
1. A compound of Formula I: ##STR00055## or a
pharmaceutically-acceptable salt thereof; wherein: Y is CR.sub.5; Z
is NR.sub.3R.sub.4, halo, H, OH, alkyl, alkyloxy, or haloalkyl;
R.sub.1a is phenyl substituted with --OH, wherein said phenyl is
additionally substituted with at least one of --OH, --CN, NO.sub.2,
C(.dbd.O)OH, --C(.dbd.O)O-alkyl, (C.sub.1-C.sub.4)alkyl, halo,
haloalkyl or haloaryl; R.sub.2 is C.sub.1-C.sub.6 alkyl or
C.sub.4-C.sub.7 cycloalkyl, wherein said alkyl or said cycloalkyl
is optionally substituted with mono- or di-alkoxy, mono- or
di-halophenyl, mono- or di-(C.sub.1-4)alkoxy phenyl, mono- or
di-(C.sub.1-4)alkyl phenyl, perhalo(C.sub.1-4)alkyl phenyl,
carboxyl, tert-butyl carboxyl, phosphoryl, (C.sub.1-6)alkyl,
(C.sub.4-7)cycloalkyl, indolyl, isoindolyl, pyridyl, naphthyl,
pyrrolyl, imidazolyl, pyrazolyl, pyrimidinyl, pyrazinyl,
pyridazinyl, furyl, thienyl, or alkylmorpholino; R.sub.3 and
R.sub.4 are independently H, C.sub.1-C.sub.6 alkyl,
tert-butyloxycarbonyl (t-Boc), morpholino(C.sub.1-C.sub.4)alkyl,
carboxy(C.sub.1-C.sub.3)alkyl,
(C.sub.1-C.sub.4)alkoxycarbonyl(C.sub.1-C.sub.3)alkyl, aryl,
heteroaryl, aryloxy, heterocyclyl, cycloalkyl, alkenyl with the
proviso that the double bond of the alkenyl is not present at the
carbon atom that is directly linked to N, alkynyl with the proviso
that the triple bond of the alkynyl is not present at the carbon
atom that is directly linked to N, perfluoroalkyl,
--S(O).sub.2alkyl, --S(O).sub.2aryl, --(C.dbd.O)heteroaryl,
--(C.dbd.O)aryl, --(C.dbd.O)(C.sub.1-C.sub.6)alkyl,
--(C.dbd.O)cycloalkyl, --(C.dbd.O)heterocyclyl, alkyl-heterocyclyl,
aralkyl, arylalkenyl, --CON R.sub.6R.sub.7,
--SO.sub.2R.sub.6R.sub.7, arylalkoxyalkyl, arylalkylalkoxy,
heteroarylalkylalkoxy, aryloxyalkyl, heteroaryloxyalkyl,
aryloxyaryl, aryloxyheteroaryl, alkylaryloxyaryl,
alkylaryloxyheteroaryl, alkylaryloxyalkylamine, alkoxycarbonyl,
aryloxycarbonyl, or heteroaryloxycarbonyl; R.sub.5 is H, --OH,
halo, optionally monosubstituted (C.sub.1-C.sub.6)alkyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkoxycarbonyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkanoyl, carbamoyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkyl carbamoyl, phenyl,
halophenyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkylphenyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkoxyphenyl, or optionally monosubstituted
perhalo(C.sub.1-C.sub.4)alkylphenyl, wherein said optional
substitution is (C.sub.1-C.sub.4)alkyl, OH, or halogen; R.sub.6 and
R.sub.7 are independently H, alkyl, aryl, heteroaryl, alkylaryl,
arylalkyl, heteroarylalkyl, or alkylheteroaryl.
2. A compound according to claim 1, wherein R.sub.1a is phenyl
substituted with mono, di, or tri-OH and further substituted with a
halo.
3. A compound according to claim 2, wherein said halo is F.
4. A compound according to claim 1, wherein R.sub.3 and R.sub.4 are
H.
5. A compound according to claim 1, wherein R.sub.5 is H.
6. A compound according to claim 1, wherein R.sub.6 is H and
R.sub.7 is methyl.
7. A compound according to claim 1, wherein, independently,
R.sub.1a is phenyl substituted at a meta position with --OH,
--CH.sub.3, tert-butyl, --CF.sub.3 or halo.
8. A compound according to claim 1, wherein, independently,
R.sub.1a is phenyl substituted at a meta position with halo,
(C.sub.1-C.sub.4)alkyl, haloalkyl, haloaryl, CN, NO.sub.2,
--C(.dbd.O)OH, or --C(.dbd.O)O-alkyl.
9. A compound according to claim 1, wherein Z is F, Br, Cl, or
I.
10. A compound selected from the group consisting of:
3-(4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
[5-(3-amino-phenyl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-a-
mine;
3-(4-benzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol-
;
3-(4-dibenzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrimidin-7-yl]-propi-
onic acid tert-butyl ester;
3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrimidin-7-yl]-propi-
onic acid;
3-bromo-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-
-5-yl)-phenol;
3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-ph-
enol;
3-tert-butyl-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-
-5-yl)-phenol;
3-(7-Isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-trifluoro-
methyl-phenol;
3-bromo-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-phenol;
3-tert-butyl-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phe-
nol; and
3-(4-Chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-triflu-
oromethyl-phenol or a pharmaceutically-acceptable salt thereof.
11. A pharmaceutical composition, comprising: a pharmaceutically
acceptable carrier, and a compound according to claim 1.
12. A pharmaceutical composition according to claim 11, further
comprising at least one anthracycline.
13. A pharmaceutical composition according to claim 12, wherein
said anthracycline is daunorubicin, doxorubicin, epirubicin,
idarubicin, or a mixture thereof.
14. A compound according to claim 1, wherein R.sub.1a is
meta-hydroxyphenyl, wherein said phenyl is additionally substituted
with at least one of OH, --CN, NO.sub.2, --C(.dbd.O)OH,
--C(.dbd.O)O-alkyl, (C.sub.1-C.sub.4)alkyl, halo, haloalkyl or
haloaryl.
Description
FIELD
This invention generally relates to pyrazolo pyrimidine
derivatives, including derivatives and analogs of inhibitors of
short chain dehydrogenase/reductase (SDR) family of NAD(P)(H)
dependent oxido-reductases. More specifically, the invention
relates to pyrazolo and pyrollo pyrimidine derivatives, including
derivatives and analogs of SDR inhibitors, pharmaceutical
compositions containing the pyrazolo pyrimidine derivatives, and
methods of making and methods of use thereof.
BACKGROUND
Cancer of the lung and bronchus (lung cancer) is the second most
common cancer among both men and women and is the leading cause of
cancer death in both sexes. Among men, age-adjusted lung cancer
incidence rates (per 100,000) range from a low of about 14 to a
high of 117, an eight- fold difference, depending upon ethnicity.
The rates among men are about two to three times greater than the
rates among women in each of the racial/ethnic groups.
Leukemia and lymphoma are the most common fatal cancers in young
men under age 39. Leukemia, Hodgkin and non-Hodgkin lymphoma and
myeloma are cancers that originate in the bone marrow or lymphatic
tissues. An estimated 106,300 people in the United States will be
diagnosed with leukemia, lymphoma or myeloma in 2002. New cases of
leukemia, Hodgkin and non-Hodgkin lymphoma and myeloma account for
8.3 percent of the 1,284,900 new cancer cases diagnosed in the
United States this year. See Surveillance, Epidemiology and End
Results (SEER) Program 1979-1998, National Cancer Institute;
American Cancer Society.
An estimated 616,695 Americans are currently living with leukemia,
Hodgkin and non-Hodgkin lymphoma and myeloma. Leukemia, lymphoma
and myeloma will cause the deaths of an estimated 58,300 people in
the United States this year. These blood cancers will account for
nearly 10.5 percent of the deaths from cancer in 2002 based on the
total of 555,500 cancer-related deaths (all sites).
The short chain dehydrogenase/reductase (SDR) family of NAD(P)(H)
dependent oxido-reductases are believed to have a role in disease,
for example, cancer, inflammatory disease, and diabetes. The SDR
family represents a diverse family of >63 human proteins
(Oppermann, U. C., et al., Chem Biol Interact, 130-132: 699-705,
2001. Kallberg, Y., et al., Eur J Biochem, 269: 4409-17, 2002.
Kallberg, Y., et al., Protein Sci, 11: 636-41, 2002). These enzymes
are responsible for the oxidation or reduction of a wide range of
endogenous (prostaglandins, steroid hormones, retinal,
dihydropteridin, UDP, and trans 2-enoyl CoA) and exogenous
chemicals (anthracyclin drugs, quininones, and others). The SDR
family members thus control the cell specific
production/destruction of potent hormones as well as the
detoxification of important classes of drugs such as the
anti-cancer agent adriamycin (Forrest, G. L. et al., Chem Biol
Interact, 129: 21-40, 2000).
Carbonyl reductase (CBR) (NADPH: secondary-alcohol oxidoreductase)
is part of a group of NADPH-dependent cytosolic enzymes called
short chain dehydrogenase/reductase (SDR) that catalyze the
reduction of various carbonyl compounds to their corresponding
alcohols. The enzyme is ubiquitous in nature and acts on a large
number of biologically and pharmacologically active compounds.
Carbonyl reductase is believed to function physiologically as a
dehydrogenase or reductase of prostaglandins or hydroxysteroids, as
well as in drug metabolism.
Carbonyl reductase is primarily monomeric in structure, and has
been characterized in humans from placenta, liver, and breast
tissue. CBR bears a low overall degree of homology (24-36%) with
other SDR enzymes from mammalian sources such as mouse and pig
(Nakanishi, M. et al. Biochem. Biophys. Acta 194: 1311-16, 193).
However, all of these enzymes are linked by two common consensus
sequences; the sequence TGxxxGxG, found in the N-terminal portion
of the molecule and responsible for binding the NADPH co-enzyme,
and the sequence YxxxK, located close to the C-terminal end of the
molecule, and active in carbonyl reduction. Differences in amino
acid sequences between these enzymes can be responsible, in part,
for differences in their respective substrate specificities for
various carbonyl compounds.
The bioreduction of prostaglandin (PGE) by carbonyl reductase
serves to regulate cellular levels of PGE. A wide variety of
biological activities are ascribed to PGEs including smooth muscle
contraction, platelet aggregation, inflammation, inhibition of
insulin secretion, and lymphocyte function. Excessive PGE
production is associated with inflammatory diseases, diabetes, and
suppression of the immune response. Inhibitors of PGE biosynthesis,
such as indomethacin and ibuprofen, are commonly used to treat
inflammation and inflammatory diseases and depressed cellular
immunity in patients with conditions such as Hodgkin's disease
(Isselbacher K. J. et al. Harrison's Principles of Internal
Medicine, Vol. 1: 431-435, 1994, McGraw-Hill, New York City).
In human liver, carbonyl reductase also reduces quinones, an
important class of mutagens and carcinogens, and appears to be the
principle mechanism for detoxification of these compounds. CBR
production is stimulated by carcinogens such as butyl
hydroxyanisole and beta-naphthoflavone that also induce other
cancer-protective enzymes (Forrest, G. L. et al. Biochim. Biophys.
Acta 1048: 149-55, 1990).
Human carbonyl reductase 1 (CBR1) has been characterized as having
similarity to carbonyl reductases from porcine lung (GI 416425),
mouse adipocytes (GI 50004), and human liver (GI 118519). Human
CBR1 is 85% identical to porcine carbonyl reductase. The role of
carbonyl reductase in cells is not understood.
Carbonyl reductase is also involved in the metabolism of
anthracyclines, a widely used class of anticancer chemotherapeutic
drugs. Daunorubicin (DNR) and Doxorubicin (DXR), the two principle
anthracyclines used in cancer chemotherapy, are reduced to their
respective alcohols by carbonyl reductase. The alcohol products are
much less effective antitumor agents than the parent compounds. In
fact, increased carbonyl reductase levels associated with some
anthracycline resistant tumors suggest that increased carbonyl
reductase activity may be responsible for drug resistance in these
cells (Soldan, M. et al. Biochem. Pharmacol. 51: 116-23, 1996;
Gonzalez, B. et al. Cancer Res. 55: 4646-50, 1995). Another problem
of anthracyclines is cardiotoxicity, but the causative agents are
suggested to be the alcohol products of carbonyl reductase
catalyzed reaction. (Forrest, G. L. et al., Chem Biol Interact,
129: 21-40, 2000.)
Daunorubicin is one of the family of anthracycline antibiotic drugs
that include daunorubicin, doxorubicin, epirubicin, and idarubicin.
These drugs are used in the treatment of acute leukemia, lymphomas,
and myeloma. Daunorubicin is used to treat acute myeloid leukemia,
acute lymphocytic leukemia, chronic myelogenous leukemia,
neuroblastoma. Liposomal daunorubicin belongs to the general group
of medicines known as antineoplastics. It is used to treat advanced
acquired immunodeficiency syndrome (AIDS)-associated Kaposi's
sarcoma (KS),
Molecules that inhibit short chain dehydrogenase/reductase (SDR)
family of NAD(P)(H) dependent oxido-reductases, for example,
carbonyl reductase, satisfy a need in the art by providing new
diagnostic or therapeutic compositions useful in the prevention and
treatment of inflammation, immunological disorders, cancer, and
drug resistance in cancerous cells, and reducing toxicity
associated with CBR catalyzed metabolites of known drugs.
SUMMARY
The invention is generally related to inhibitors of short chain
dehydrogenase/reductase (SDR) family of NAD(P)(H) dependent
oxido-reductases, and derivatives and analogs thereof. The
invention further relates to pharmaceutical compositions containing
the inhibitors of SDR family of NAD(P)(H) dependent
oxido-reductases, and derivatives and analogs thereof, methods of
making the inhibitors of SDR family of NAD(P)(H) dependent
oxido-reductases and derivatives and analogs thereof, and methods
of use thereof.
In one embodiment, the present invention is directed to a compound
of Formula I or II:
##STR00002## or a pharmaceutically-acceptable salt or prodrug
thereof; wherein: Y is N or CR.sub.5; Z is NR.sub.3R.sub.4, halo,
H, ,OH, alkyl, alkyloxy, or haloalkyl; R.sub.1a is indolyl,
thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl,
wherein said phenyl is substituted with at least one of OH,
--NR.sub.3R.sub.4, --C(.dbd.O)NR.sub.6R.sub.7, --CN,
NO.sub.2--C(.dbd.O)OH, --C(.dbd.O)O-alkyl, (C.sub.1-C.sub.4)alkyl,
halo, haloalkyl or haloaryl; and wherein said indolyl, thiazolyl,
benzyl, biphenylyl, thiophenyl, or pyrrolyl is optionally
substituted with OH, --NR.sub.3R.sub.4, --C(.dbd.O)NR.sub.6R.sub.7,
--CN, NO.sub.2, --C(.dbd.O)O--R.sub.3, (C.sub.1-C.sub.4)alkyl,
halo, haloalkyl or haloaryl; R.sub.1b is indolyl, thiazolyl,
benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl wherein said
indoyl, thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, phenyl
is optionally substituted with --OH, --NR.sub.3R.sub.4,
--C(.dbd.O)NR.sub.6R.sub.7, --CN, NO.sub.2, --C(.dbd.O)O--R.sub.3,
(C.sub.1-C.sub.4)alkyl, halo, haloalkyl, or haloaryl; R.sub.2 is
C.sub.1-C.sub.6 alkyl or C.sub.4-C.sub.7 cycloalkyl, wherein said
alkyl or said cycloalkyl is optionally substituted with mono- or
di-alkoxy, mono- or di-halophenyl, mono- or di-(C.sub.1-4)alkoxy
phenyl, mono- or di-(C.sub.1-4)alkyl phenyl,
perhalo(C.sub.1-4)alkyl phenyl, carboxyl, tert-butyl carboxyl,
phosphoryl, (C.sub.1-6)alkyl, (C.sub.4-7)cycloalkyl, indolyl,
isoindolyl, pyridyl, naphthyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, furyl, thienyl, or
alkylmorpholino; R.sub.3 and R.sub.4 are independently H,
C.sub.1-C.sub.6 alkyl, t-Boc, morpholino(C.sub.1-C.sub.4)alkyl,
carboxy(C.sub.1-C.sub.3)alkyl,
(C.sub.1-C.sub.4)alkoxycarbonyl(C.sub.1-C.sub.3)alkyl, aryl,
heteroaryl, aryloxy, heterocycle, cycloalkyl, alkenyl with the
proviso that the double bond of the alkenyl is not present at the
carbon atom that is directly linked to N, alkynyl with the proviso
that the triple bond of the alkynyl is not present at the carbon
atom that is directly linked to N, perfluoroalkyl,
--S(O).sub.2alkyl, --S(O).sub.2aryl, --(C.dbd.O)heteroaryl,
--(C.dbd.O)aryl, --(C.dbd.O)(C.sub.1-C.sub.6) alkyl,
--(C.dbd.O)cycloalkyl, --(C.dbd.O)heterocycle, alkyl-heterocycle,
aralkyl, arylalkenyl, --CON R.sub.6R.sub.7,
--SO.sub.2R.sub.6R.sub.7, arylalkoxyalkyl, arylalkylalkoxy,
heteroarylalkylalkoxy, aryloxyalkyl, heteroaryloxyalkyl,
aryloxyaryl, aryloxyheteroaryl, alkylaryloxyaryl,
alkylaryloxyheteroaryl, alkylaryloxyalkyamine, alkoxycarbonyl,
aryloxycarbonyl, or heteroaryloxycarbonyl; R.sub.5 are
independently H, --OH, halo, optionally monosubstituted
(C.sub.1-C.sub.6)alkyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkoxycarbonyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkanoyl, carbamoyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkyl carbamoyl, phenyl, halophenyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkylphenyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkoxyphenyl, or optionally
monosubstituted perhalo(C.sub.1-C.sub.4)alkylphenyl, wherein said
optional substitution is (C.sub.1-C.sub.4)alkyl, OH, or halogen;
R.sub.6 and R.sub.7 are independently H, alkyl, aryl, heteroaryl,
alkylaryl, arylalkyl, heteroarylalkyl, or alkylheteroaryl; provided
the compound is not
1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.
In another embodiment, the present invention is directed to a
method for preventing or treating cancer in a mammal, comprising
the step of administering to the mammal an effective amount of a
compound of formula I or formula II, or a pharmaceutically
acceptable salt thereof.
In yet another embodiment, the present invention is directed to a
method for preventing or treating a disease or condition associated
with carbonyl reductase 1 in a mammal in need thereof, comprising
the steps of administering to the mammal a composition comprising
an effective amount of a compound of formula I or formula II, or a
pharmaceutically acceptable salt thereof.
In yet another embodiment, the present invention is directed to a
method of preventing or treating a disease or condition associated
with the synthesis of prostaglandin E in a mammal in need thereof
comprising the steps of administering to the mammal a composition
comprising an effective amount of a compound of formula I or
formula II, or a pharmaceutically acceptable salt thereof, and
inhibiting synthesis of prostaglandin E2.
In a further embodiment, the present invention is directed to a
method for preventing or treating a disease or condition associated
with short chain dehydrogenase/reductase (SDR) family of NAD(P)(H)
dependent oxido-reductases in a mammal in need thereof, comprising
the step of administering to the mammal an effective amount of a
composition comprising an effective amount of a compound of formula
I or formula II, or a pharmaceutically acceptable salt thereof.
In a further embodiment, the present invention is directed to a
method for identifying a therapeutic cancer treatment, comprising
the steps of contacting a tumor cell culture with an effective
amount of a composition comprising an effective amount of a
compound of formula I or formula II, or a pharmaceutically
acceptable salt thereof, measuring growth inhibition of the tumor
cells in culture; and identifying a therapeutic cancer treatment
for a mammalian subject by inhibition of the tumor cell growth in
culture.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the role of CBR in the biosynthesis of
prostaglandins.
FIG. 2 shows enzymatic activity of 11.beta.-hydroxysteroid
dehydrogenase I and 11.beta.-hydroxysteroid dehydrogenase II.
FIG. 3 shows enzymatic activity of carbonyl reductase 1 (CBR1) on
daunorubicin.
FIG. 4 shows carbonyl reductase 1 (CBR1) is up-regulated in some
classes of lung tumors.
FIG. 5 shows AB129 inhibits menadione reducing activity of carbonyl
reductase 1 (CBR1).
FIG. 6 shows kinetic analysis of carbonyl reductase 1 (CBR1).
FIG. 7 shows selection of RNAi hairpins for targeting of carbonyl
reductase 1 (CBR1).
FIG. 8 shows carbonyl reductase 1 (CBR1) is required for A549 lung
carcinoma cell viability.
FIG. 9 shows the structure of glycyrrizic acid.
FIG. 10 shows the structure of (1) AB129, (2) PP1, (3) AB60, and
(4) AB61.
FIG. 11 shows that AB129 causes a morphological change in human
lung carcinoma cells.
FIG. 12 shows effects of AB129 on cell cycle of A549 lung carcinoma
cells.
FIG. 13 shows probes for an affinity experiment to identify AB129
target.
FIG. 14 shows an affinity experiment using A549 lung carcinoma cell
lysate.
FIG. 15 shows protein identification by collision induced
dissociation mass spectrometry (LC/MS/MS).
FIG. 16 shows amino acid sequence identification of trypsin digest
by mass spectrometry.
FIG. 17 shows mass spectrometric identification of protein
fragments.
FIG. 18 shows mass spectrometric identification of protein
fragments.
FIG. 19 shows mass spectrometric identification of protein
fragments.
FIG. 20 shows mass spectrometric identification of protein
fragments.
FIG. 21 shows mass spectrometric identification of protein
fragments.
FIG. 22 shows mass spectrometric identification of protein
fragments.
FIG. 23 kinetic parameters of AB129 inhibition of carbonyl
reductase 1 (CBR1).
FIG. 24 shows a molecular model for docking of AB129 within porcine
carbonyl reductase 1 (CBR1).
FIG. 25 shows conserved amino acid residues in short chain
dehydrogenase/reductase (SDR) enzymes.
FIG. 26 shows sequence alignment of NADPH binding pocket in SDR
enzymes.
FIG. 27 shows a Rossman fold of SDR enzymes.
FIG. 28 shows effects on inhibition by mutation at Asn90 of
carbonyl reductase 1 (CBR1).
FIG. 29 shows Asn90 has a role for binding of AB129 to carbonyl
reductase 1 (CBR1).
FIG. 30 shows N90V mutant of carbonyl reductase 1 (CBR1) is less
sensitive to AB129 than wild type CBR1.
FIG. 31 shows glutathione-modified eicosanoids.
FIG. 32 shows glutathione binding activity of wild type and mutant
carbonyl reductase 1 (CBR1).
FIG. 33 shows carbonyl reductase (CBR) can cause anthracycline
resistance.
FIG. 34 shows that AB129 reinforces the cytotoxicity of
daunorubicin.
FIG. 35 shows AB129 analogs display differing selectivities for
carbonyl reductase (CBR) and kinases.
FIG. 36 shows potential library substituents for inhibitors of SDR
enzymes.
FIG. 37 shows pyrrolopyrimidine scaffold validation.
FIG. 38 shows solid phase pyrrolopyrimidine library synthesis.
FIG. 39 shows scaffold loading optimization.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
With respect to pyrazolo pyrimidine or, "derivative" refers to a
compound of general formula I or II:
##STR00003## where the variables are as defined herein.
With respect to pyrazolo pyrimidine or AB-129 compound, "analog" or
"functional analog" refers to a modified form of the respective
pyrazolo pyrimidine or AB-129 compound derivative in which one or
more chemically derivatized functional substituent (R.sub.1a,
R.sub.1b, R.sub.2, R.sub.3, R.sub.4 or Z) or a ring atom (Y) has
been modified such that the analog retains substantially the same
biological activity or improved biological activity as the
unmodified pyrazolo pyrimidine or AB-129 compound derivative in
vivo and/or in vitro.
The present invention is directed to pyrazolo pyrimidine
derivatives, compositions containing these derivatives, and methods
of their use for the prevention and treatment of, inter alia,
cancer, metastatic cancer, inflammation, and diabetes.
The following definitions are provided for the full understanding
of terms and abbreviations used in this specification.
As used herein and in the appended claims, the singular forms "a,"
"an," and "the" include the plural reference unless the context
clearly indicates otherwise. Thus, for example, a reference to "an
antagonist" or "an agonist" includes a plurality of such
antagonists or a plurality of such agonists, and a reference to "a
compound" is a reference to one or more compounds and equivalents
thereof known to those skilled in the art, and so forth.
The abbreviations in the specification correspond to units of
measure, techniques, properties, or compounds as follows: "min"
means minutes, "h" means hour(s), ".mu.L" means microliter(s), "mL"
means milliliter(s), "mM" means millimolar, "M" means molar,
"mmole" means millimole(s), "cm" means centimeters, "SEM" means
standard error of the mean and "IU" means International Units,
".degree. C." means degrees Celcius. ".DELTA.ED.sub.50 value" means
dose which results in 50% alleviation of the observed condition or
effect (50% mean maximum endpoint), ".DELTA.ID.sub.50" means dose
which results in 50% inhibition of an observed condition or effect
or biochemical process (50% mean maximum endpoint).
"Alkyl" refers to an optionally substituted, saturated straight,
branched, or cyclic hydrocarbon having from about 1 to about 20
carbon atoms (and all combinations and subcombinations of ranges
and specific numbers of carbon atoms therein), with from about 1 to
about 8 carbon atoms, herein referred to as "lower alkyl", being
preferred. Alkyl groups include, but are not limited to, methyl,
ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl,
cyclopentyl, isopentyl, neopentyl, n-hexyl, isohexyl, cyclohexyl,
cyclooctyl, adamantyl, 3-methylpentyl, 2,2-dimethylbutyl, and
2,3-dimethylbutyl.Lower alkyl refers to alkyl having 1 to 4 carbon
atoms.
"Cycloalkyl" refers to an optionally substituted, alkyl group
having one or more rings in their structures having from about 3 to
about 20 carbon atoms (and all combinations and subcombinations of
ranges and specific numbers of carbon atoms therein), with from
about 3 to about 10 carbon atoms being preferred. Multi-ring
structures can be bridged or fused ring structures. Groups include,
but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,
cyclohexyl, cyclooctyl, and adamantyl. Specifically included within
the definition of "cyclic alkyl" are those aliphatic hydrocarbon
chains that are optionally substituted.
"Perfluorinated alkyl" refers to an alkyl, as defined above, in
which the hydrogens directly attached to the carbon atoms are
completely replaced by fluorine.
"Alkenyl" refers to an alkyl group of at least two carbon atoms
having one or more double bonds, wherein alkyl is as defined
herein. Alkenyl groups can be optionally substituted.
"Alkynyl" refers to an alkyl group of at least two carbon atoms
having one or more triple bonds, wherein alkyl is as defined
herein. Alkynyl groups can be optionally substituted.
"Aryl" as used herein, refers to an optionally substituted, mono-,
di-, tri-, or other multicyclic aromatic ring system having from
about 5 to about 50 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein), with from about 6 to about 10 carbons being preferred.
Non-limiting examples include, for example, phenyl, naphthyl,
anthracenyl, and phenanthrenyl.
"Heteroaryl" refers to an optionally substituted, mono-, di-, tri-,
or other multicyclic aromatic ring system that includes at least
one, and preferably from 1 to about 4 sulfur, oxygen, or nitrogen
heteroatom ring members. Heteroaryl groups can have, for example,
from about 3 to about 50 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein), with from about 4 to about 10 carbons being preferred.
Non-limiting examples of heteroaryl groups include, for example,
pyrryl, furyl, pyridyl, 1,2,4-thiadiazolyl, pyrimidyl, thienyl,
isothiazolyl, imidazolyl, tetrazolyl, pyrazinyl, pyrimidyl,
quinolyl, isoquinolyl, thiophenyl, benzothienyl, isobenzofuryl,
pyrazolyl, indolyl, purinyl, carbazolyl, benzimidazolyl, and
isoxazolyl.
"Heterocyclic ring" refers to a stable 5- to 7-membered monocyclic
or bicyclic or 7- to 10-membered bicyclic heterocyclic ring that is
saturated, partially unsaturated or unsaturated (aromatic), and
which contains carbon atoms and from 1 to 4 heteroatoms
independently selected from the group consisting of N, O and S and
including any bicyclic group in which any of the above defined
heterocyclic rings is fused to a benzene ring. The nitrogen and
sulfur heteroatoms may optionally be oxidized. The heterocyclic
ring may be attached to its pendant group at any heteroatom or
carbon atom that results in a stable structure. The heterocyclic
rings described herein may be substituted on carbon or on a
nitrogen atom if the resulting compound is stable. If specifically
noted, a nitrogen atom in the heterocycle may optionally be
quaternized. It is preferred that when the total number of S and O
atoms in the heterocycle exceeds one, then these heteroatoms are
not adjacent to one another. It is preferred that the total number
of S and O atoms in the heterocycle is not more than one. Examples
of heterocycles include, but are not limited to, 1H-indazole,
2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl, 3H-indolyl,
4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,
6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazalonyl, carbazolyl, 4H-carbazolyl,
.alpha.-, .beta.-, or .gamma.-carbolinyl, chromanyl, chromenyl,
cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl,
isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,
isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl., oxazolyl, oxazolidinylpyrimidinyl, phenanthridinyl,
phenanthrolinyl, phenoxazinyl, phenazinyl, phenothiazinyl,
phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,
piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,
pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, carbolinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred heterocycles
include, but are not limited to, pyridinyl, furanyl, thienyl,
pyrrolyl, pyrazolyl, imidazolyl, indolyl, benzimidazolyl,
1H-indazolyl, oxazolidinyl, benzotriazolyl, benzisoxazolyl,
oxindolyl, benzoxazolinyl, or isatinoyl. Also included are fused
ring and spiro compounds containing, for example, the above
heterocycles.
"Alkoxy" refers to the group R--O-- where R is an alkyl group as
defined herein.
"Aryloxy" refers to the group R--O-- where R is an aryl group, as
defined herein.
"Heteroaryloxy" refers to the group R--O-- where R is a heteroaryl
group, as defined herein.
"Alkanoyl" refers to the group R--C(.dbd.O) where R is an alkyl
group of 1 to 5 carbon atoms.
"Alkanoyloxy" refers to the group R--C(.dbd.O)--O where R is an
alkyl group of 1 to 5 carbon atoms.
"Halo," refers to chloro, bromo, fluoro, and iodo.
"Haloalkyl," or "haloaryl" refers to an alkyl or aryl, as defined
above, in which one or more hydrogens directly attached to the
carbon atoms are replaced by one or more halo substituents.
"Optional" or "optionally" means that the subsequently described
event or circumstance may or may not occur, and that the
description includes instances in which it does not. For example,
optionally substituted phenyl indicates either unsubstituted
phenyl, or phenyl mono-, di, or tri-substituted, independently,
with OH, COOH, lower alkyl, lower alkoxy, halo, nitro, amino,
alkylamino, dialkylamino, trifluoromethyl and/or cyano.
By "therapeutically effective dose" herein is meant a dose that
produces effects for which it is administered. The exact dose will
depend on the purpose of the treatment, and will be ascertainable
by one skilled in the art using known techniques (see, e.g.,
Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd,
1999, The Art, Science And Technology Of Pharmaceutical
Compounding; and Pickar, 1999, Dosage Calculations). "Effective
amount" refers to an amount of a compound that can be
therapeutically effective to inhibit, prevent or treat the symptoms
of particular disease, disorder or side effect.
"Pharmaceutically acceptable" refers to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for contact with the tissues of
human beings and animals without excessive toxicity, irritation,
allergic response, or other problem complications commensurate with
a reasonable benefit/risk ratio.
"In combination with", "combination therapy" and "combination
products" refer, in certain embodiments, to the concurrent
administration to a patient of a first therapeutic and the
compounds as used herein. When administered in combination, each
component can be administered at the same time or sequentially in
any order at different points in time. Thus, each component can be
administered separately but sufficiently closely in time so as to
provide the desired therapeutic effect.
"Dosage unit" refers to physically discrete units suited as unitary
dosages for the particular individual to be treated. Each unit can
contain a predetermined quantity of active compound(s) calculated
to produce the desired therapeutic effect(s) in association with
the required pharmaceutical carrier. The specification for the
dosage unit forms can be dictated by (a) the unique characteristics
of the active compound(s) and the particular therapeutic effect(s)
to be achieved, and (b) the limitations inherent in the art of
compounding such active compound(s).
"Stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate,
solvate, acid salt hydrate, N-oxide or isomorphic crystalline form
thereof" refer to derivatives of the disclosed compounds wherein
the parent compound is modified by making acid or base salts
thereof. Examples of stereoisomer, prodrug, pharmaceutically
acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or
isomorphic crystalline form thereof include, but are not limited
to, mineral or organic acid salts of basic residues such as amines;
alkali or organic salts of acidic residues such as carboxylic
acids; and the like. The stereoisomer, prodrug, pharmaceutically
acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or
isomorphic crystalline form thereof include the conventional
non-toxic salts or the quaternary ammonium salts of the parent
compound formed, for example, from non-toxic inorganic or organic
acids. For example, such conventional non-toxic salts include those
derived from inorganic acids such as hydrochloric, hydrobromic,
sulfuric, sulfamic, phosphoric, nitric and the like; and the salts
prepared from organic acids such as acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,
pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic,
salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic,
methanesulfonic, ethane disulfonic, oxalic, isethionic, and the
like. These physiologically acceptable salts are prepared by
methods known in the art, e.g., by dissolving the free amine bases
with an excess of the acid in aqueous alcohol, or neutralizing a
free carboxylic acid with an alkali metal base such as a hydroxide,
or with an amine.
Compounds described herein throughout, can be used or prepared in
alternate forms. For example, many amino-containing compounds can
be used or prepared as an acid addition salt. Often such salts
improve isolation and handling properties of the compound. For
example, depending on the reagents, reaction conditions and the
like, compounds as described herein can be used or prepared, for
example, as their hydrochloride or tosylate salts. Isomorphic
crystalline forms, all chiral and racemic forms, N-oxide, hydrates,
solvates, and acid salt hydrates, are also contemplated to be
within the scope of the present compositions and methods.
Certain acidic or basic compounds can exist as zwitterions. All
forms of the compounds, including free acid, free base and
zwitterions, are contemplated to be within the scope of the present
compositions and methods. It is well known in the art that
compounds containing both amino and carboxyl groups often exist in
equilibrium with their zwitterionic forms. Thus, any of the
compounds described herein throughout that contain, for example,
both amino and carboxyl groups, also include reference to their
corresponding zwitterions.
The term "treating" includes the administration of the compounds or
agents of the present invention to prevent or delay the onset of
the symptoms, complications, or biochemical indicia of a disease,
alleviating the symptoms or arresting or inhibiting further
development of the disease, condition, or disorder (e.g., cancer).
Treatment may be prophylactic (to prevent or delay the onset of the
disease, or to prevent the manifestation of clinical or subclinical
symptoms thereof) or therapeutic suppression or alleviation of
symptoms after the manifestation of the disease.
In general, the phrase "well tolerated" refers to the absence of
adverse changes in health status that occur as a result of the
treatment and would affect treatment decisions. Except when noted,
the terms "patient" or "subject" are used interchangeably and refer
to mammals such as human patients and non-human primates, as well
as experimental animals such as rabbits, rats, and mice, and other
animals.
"Prodrug" refers to compounds specifically designed to maximize the
amount of active species that reaches the desired site of reaction
which are of themselves typically inactive or minimally active for
the activity desired, but through biotransformation are converted
into biologically active metabolites.
"Stereoisomers" refers to compounds that have identical chemical
constitution, but differ as regards the arrangement of the atoms or
groups in space.
When any variable occurs more than one time in any constituent or
in any formula, its definition in each occurrence is independent of
its definition at every other occurrence. Combinations of
substituents and/or variables are permissible only if such
combinations result in stable compounds.
"Short chain dehydrogenase reductase (SDR)" refers to a family of
NAD(P)(H) dependent oxido-reductases represent a diverse family of
greater than 63 human proteins. These enzymes are responsible for
the oxidation or reduction of a wide range of endogenous
(prostaglandins, steroid hormones, retinal, dihydropteridin, UDP,
and trans 2-enoyl CoA) and exogenous chemicals (e.g., anthracyclin
drugs).
"Modulate" refers to the suppression, enhancement or induction of a
function or condition. For example, the pyrazolo pyrimidine or
AB-129 compounds, derivatives and analogs thereof of the invention
can modulate cancer by inhibition of short chain dehydrogenase
reductase (SDR) enzyme activity. For example, AB-129 compounds,
derivatives and analogs thereof can inhibit carbonyl reductase 1
(CBR1) activity in lung carcinoma cells thereby alleviating lung
cancer by inhibiting or reducing growth of lung carcinoma
cells.
"Carbonyl reductase" refers to a family of enzymes, for example,
carbonyl reductase 1 (NADPH: secondary-alcohol oxidoreductase)
which is part of a group of NADPH-dependent cytosolic enzymes
called short chain dehydrogenase/reductase (SDR) that catalyze the
reduction of various carbonyl compounds to their corresponding
alcohols. The enzyme is ubiquitous in nature and acts on a large
number of biologically and pharmacologically active compounds.
Carbonyl reductase is believed to function physiologically as
dehydrogenases of prostaglandins or hydroxysteroids, as well as in
drug metabolism.
"11.beta.-hydroxysteroid dehydrogenase (11.beta.-HSD)" refers to
11.beta.-hydroxysteroid dehydrogenase I or 17.beta.-hydroxysteroid
dehydrogenase II, or an enzyme with a related activity.
"17.beta.-hydroxysteroid dehydrogenase(17.beta.-HSD)" refers to
17.beta.-hydroxysteroid dehydrogenase I or 17.beta.-hydroxysteroid
dehydrogenase II, or an enzyme with a related activity.
"Anthracycline anti-cancer agent" refers to the family of
anthracycline antibiotic drugs that include, for example
daunorubicin, doxorubicin, epirubicin, and idarubicin.
"Cardiotoxic side effect" refers to acute or chronic cardiomyopathy
that can lead to congestive heart failure. The development of
chronic cardiotoxicity is clinically important. Chronic
cardiotoxicity can develop many years after treatment with
anthracyclines. Children and younger adults treated with
anthracyclines are exposed to a lifetime risk of developing serious
cardiomyopathy. Because cancer patients are not usually monitored
for more than 5-7 years, the number of these patients developing
late-onset cardiomyopathies can be expected to increase
substantially in the future.
"Cancer" or "malignancy" are used as synonymous terms and refer to
any of a number of diseases that are characterized by uncontrolled,
abnormal proliferation of cells, the ability of affected cells to
spread locally or through the bloodstream and lymphatic system to
other parts of the body (i.e., metastasize) as well as any of a
number of characteristic structural and/or molecular features. A
"cancerous" or "malignant cell" is understood as a cell having
specific structural properties, lacking differentiation and being
capable of invasion and metastasis. Examples of cancers are kidney,
colon, breast, prostate and liver cancer. (see DeVita, V. et al.
(eds.), 2001, CANCER PRINCIPLES AND PRACTICE OF ONCOLOGY, 6th. Ed.,
Lippincott Williams & Wilkins, Philadelphia, Pa.; this
reference is herein incorporated by reference in its entirety for
all purposes).
"Cancer-associated" refers to the relationship of a nucleic acids
and its expression, or lack thereof, or a protein and its level or
activity, or lack thereof, to the onset of malignancy in a subject
cell. For example, cancer can be associated with expression of a
particular gene that is not expressed, or is expressed at a lower
level, in a normal healthy cell. Conversely, a cancer-associated
gene can be one that is not expressed in a malignant cell (or in a
cell undergoing transformation), or is expressed at a lower level
in the malignant cell than it is expressed in a normal healthy
cell.
"Neoplastic cells" and "neoplasia" refer to cells which exhibit
relatively autonomous growth, so that they exhibit an aberrant
growth phenotype characterized by a significant loss of control of
cell proliferation. Neoplastic cells comprise cells which can be
actively replicating or in a temporary non-replicative resting
state (G1 or G0); similarly, neoplastic cells can comprise cells
which have a well-differentiated phenotype, a poorly-differentiated
phenotype, or a mixture of both type of cells. Thus, not all
neoplastic cells are necessarily replicating cells at a given
timepoint. The set defined as neoplastic cells consists of cells in
benign neoplasms and cells in malignant (or frank) neoplasms.
Frankly neoplastic cells are frequently referred to as cancer
(discussed supra), typically termed carcinoma if originating from
cells of endodermal or ectodermal histological origin, or sarcoma
if originating from cell types derived from mesoderm.
In the context of the invention, the term "transformation" refers
to the change that a normal cell undergoes as it becomes malignant.
In eukaryotes, the term "transformation" can be used to describe
the conversion of normal cells to malignant cells in cell
culture.
"Proliferating cells" are those which are actively undergoing cell
division and growing exponentially.
"Loss of cell proliferation control" refers to the property of
cells that have lost the cell cycle controls that normally ensure
appropriate restriction of cell division. Cells that have lost such
controls proliferate at a faster than normal rate, without
stimulatory signals, and do not respond to inhibitory signals.
"Leukemia" refers to cancer of cells in the bloodstream or
lymphatic system. Types of leukemia include but are not limited to,
Acute Lymphoblastic Leukemia (Adult or Childhood), Acute Myeloid
Leukemia (Adult or Childhood), Chronic Lymphocytic Leukemia,
Chronic Myelogenous Leukemia, or Hairy Cell Leukemia.
"Kaposi's sarcoma (KS)" refers to a sarcoma that develops in
connective tissues such as cartilage, bone, fat, muscle, blood
vessels, or fibrous tissues (related to tendons or ligaments). The
vast majority of KS cases have developed in association with human
immunodeficiency virus (HIV) infection and the acquired
immunodeficiency syndrome (AIDS). KS tumors develop in the tissues
below the skin surface, or in the mucous membranes of the mouth,
nose, or anus.
"Inflammation" or "inflammatory response" refers to an innate
immune response that occurs when tissues are injured by bacteria,
trauma, toxins, heat, or any other cause. The damaged tissue
releases compounds including histamine, bradykinin, and serotonin.
Inflammation refers to both acute responses (i.e., responses in
which the inflammatory processes are active) and chronic responses
(i.e., responses marked by slow progression and formation of new
connective tissue). Acute and chronic inflammation can be
distinguished by the cell types involved. Acute inflammation often
involves polymorphonuclear neutrophils; whereas chronic
inflammation is normally characterized by a lymphohistiocytic
and/or granulomatous response. Inflammation includes reactions of
both the specific and non-specific defense systems. A specific
defense system reaction is a specific immune system reaction
response to an antigen (possibly including an autoantigen). A
non-specific defense system reaction is an inflammatory response
mediated by leukocytes incapable of immunological memory. Such
cells include granulocytes, macrophages, neutrophils and
eosinophils. Examples of specific types of inflammation are diffuse
inflammation, focal inflammation, croupous inflammation,
interstitial inflammation, obliterative inflammation,
parenchymatous inflammation, reactive inflammation, specific
inflammation, toxic inflammation and traumatic inflammation.
"Diabetes mellitus" or "diabetes" refers to a disease or condition
that is generally characterized by metabolic defects in production
and utilization of glucose which result in the failure to maintain
appropriate blood sugar levels in the body. The result of these
defects is elevated blood glucose, referred to as "hyperglycemia."
Two major forms of diabetes are Type 1 diabetes and Type 2
diabetes. As described above, Type 1 diabetes is generally the
result of an absolute deficiency of insulin, the hormone which
regulates glucose utilization. Type 2 diabetes often occurs in the
face of normal, or even elevated levels of insulin and can result
from the inability of tissues to respond appropriately to insulin.
Most Type 2 diabetic patients are insulin resistant and have a
relative deficiency of insulin, in that insulin secretion can not
compensate for the resistance of peripheral tissues to respond to
insulin. In addition, many Type 2 diabetics are obese. Other types
of disorders of glucose homeostasis include Impaired Glucose
Tolerance, which is a metabolic stage intermediate between normal
glucose homeostasis and diabetes, and Gestational Diabetes
Mellitus, which is glucose intolerance in pregnancy in women with
no previous history of Type 1 or Type 2 diabetes.
"Secondary diabetes" is diabetes resulting from other identifiable
etiologies which include: genetic defects of .beta. cell function
(e.g., maturity onset-type diabetes of youth, referred to as
"MODY," which is an early-onset form of Type 2 diabetes with
autosomal inheritance; see, e.g., Fajans S. et al., Diabet. Med. 9
Suppl 6: S90-5, 1996, and Bell, G. et al., Annu. Rev. Physiol. 58:
171-86, 1996); genetic defects in insulin action; diseases of the
exocrine pancreas (e.g., hemochromatosis, pancreatitis, and cystic
fibrosis); certain endocrine diseases in which excess hormones
interfere with insulin action (e.g., growth hormone in acromegaly
and cortisol in Cushing's syndrome); certain drugs that suppress
insulin secretion (e.g., phenytoin) or inhibit insulin action
(e.g., estrogens and glucocorticoids); and diabetes caused by
infection (e.g., rubella, Coxsackie, and CMV); as well as other
genetic syndromes.
The guidelines for diagnosis for Type 2 diabetes, impaired glucose
tolerance, and gestational diabetes have been outlined by the
American Diabetes Association (see, e.g., The Expert Committee on
the Diagnosis and Classification of Diabetes Mellitus, Diabetes
Care, 2 (Suppl 1): S5-19, 1999).
Methods of Treatment
Short chain dehydrogenases/reductases (SDRs), for example, carbonyl
reductase 1 (CBR1) have a role in metabolism and disease.
AB129-type compounds and analogs thereof are inhibitors of the SDR
enzyme family. AB129-type compounds and analogs are useful for
medical treatment, for example, cancer therapy, and have been shown
to have biological activity.
(1) Human carbonyl reductase 1 (CBR1) is within the family of short
chain dehydrogenases/reductases (SDRs).
(2) The SDR enzyme family has more than 1600 members. Greater than
63 SDR enzymes are found in human.
(3) SDR enzymes catalyze an NAD(P)(H)-dependent oxidoreduction or
dehydrogenation.
(4) The catalytic active site of the SDR enzyme comprises an
S/YxxxK catalytic triad.
(5) The function of SDR enzymes include intermediary metabolism,
lipid hormone metabolism (e.g., steroids, prostaglandins,
retinols/retinals) and enzymes of unknown function.
(6) SDR enzymes are correlated with many genetic and metabolic
disorders.
(7) SDR enzymes can regulate the nuclear hormone switch (e.g.,
cortisone, estradiol, prostaglandin) as an important regulatory
target by AB129-type compounds.
(8) AB129-type compounds and analogs thereof that inhibit carbonyl
reductase 1 (CBR1) activity are useful for treatment of lung
cancer, colon cancer, metastatic cancer, or cancer drug
resistance.
(9) AB129-type compounds and analogs thereof that inhibit
11.beta.-hydroxysteroid dehydrogenase activity and result in
decreased levels of cortisone are useful for treatment of diabetes
or obesity.
(10) AB129-type compounds and analogs thereof that inhibit
17.beta.-hydroxysteroid dehydrogenase activity are useful for
treatment of inflammatory disease, ovarian cancer or breast
cancer.
In one embodiment, the present invention is directed to a compound
of Formula I or II:
##STR00004## or a pharmaceutically-acceptable salt or prodrug
thereof; wherein: Y is N or CR.sub.5; Z is NR.sub.3R.sub.4, halo,
H, ,OH, alkyl, alkyloxy, or haloalkyl; R.sub.1a is indolyl,
thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl,
wherein said phenyl is substituted with at least one of OH,
--NR.sub.3R.sub.4, --C(.dbd.O)NR.sub.6R.sub.7, --CN,
NO.sub.2--C(.dbd.O)OH, --C(.dbd.O)O-alkyl, (C.sub.1-C.sub.4)alkyl,
halo, haloalkyl or haloaryl; and wherein said indolyl, thiazolyl,
benzyl, biphenylyl, thiophenyl, or pyrrolyl is optionally
substituted with OH, --NR.sub.3R.sub.4, --C(.dbd.O)NR.sub.6R.sub.7,
--CN, NO.sub.2, --C(.dbd.O)O--R.sub.3, (C.sub.1-C.sub.4)alkyl,
halo, haloalkyl or haloaryl; R.sub.1b is indolyl, thiazolyl,
benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl wherein said
indoyl, thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, phenyl
is optionally substituted with --OH, --NR.sub.3R.sub.4,
--C(.dbd.O)NR.sub.6R.sub.7, --CN, NO.sub.2, --C(.dbd.O)O--R.sub.3,
(C.sub.1-C.sub.4)alkyl, halo, haloalkyl, or haloaryl; R.sub.2 is
C.sub.1-C.sub.6 alkyl or C.sub.4-C.sub.7 cycloalkyl, wherein said
alkyl or said cycloalkyl is optionally substituted with mono- or
di-alkoxy, mono- or di-halophenyl, mono- or di-(C.sub.1-4)alkoxy
phenyl, mono- or di-(C.sub.1-4)alkyl phenyl,
perhalo(C.sub.1-4)alkyl phenyl, carboxyl, tert-butyl carboxyl,
phosphoryl, (C.sub.1-6)alkyl, (C.sub.4-7)cycloalkyl, indolyl,
isoindolyl, pyridyl, naphthyl, pyrrolyl, imidazolyl, pyrazolyl,
pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, furyl, thienyl, or
alkylmorpholino; R.sub.3 and R.sub.4 are independently H,
C.sub.1-C.sub.6 alkyl, t-Boc, morpholino(C.sub.1-C.sub.4)alkyl,
carboxy(C.sub.1-C.sub.3)alkyl,
(C.sub.1-C.sub.4)alkoxycarbonyl(C.sub.1-C.sub.3)alkyl, aryl,
heteroaryl, aryloxy, heterocycle, cycloalkyl, alkenyl with the
proviso that the double bond of the alkenyl is not present at the
carbon atom that is directly linked to N, alkynyl with the proviso
that the triple bond of the alkynyl is not present at the carbon
atom that is directly linked to N, perfluoroalkyl,
--S(O).sub.2alkyl, --S(O).sub.2aryl, --(C.dbd.O)heteroaryl,
--(C.dbd.O)aryl, --(C.dbd.O)(C.sub.1-C.sub.6)alkyl,
--(C.dbd.O)cycloalkyl, --(C.dbd.O)heterocycle, alkyl-heterocycle,
aralkyl, arylalkenyl, --CON R.sub.6R.sub.7,
--SO.sub.2R.sub.6R.sub.7, arylalkoxyalkyl, arylalkylalkoxy,
heteroarylalkylalkoxy, aryloxyalkyl, heteroaryloxyalkyl,
aryloxyaryl, aryloxyheteroaryl, alkylaryloxyaryl,
alkylaryloxyheteroaryl, alkylaryloxyalkyamine, alkoxycarbonyl,
aryloxycarbonyl, or heteroaryloxycarbonyl; R.sub.5 are
independently H, --OH, halo, optionally monosubstituted
(C.sub.1-C.sub.6)alkyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkoxycarbonyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkanoyl, carbamoyl, optionally monosubstituted
(C.sub.1-C.sub.4)alkyl carbamoyl, phenyl, halophenyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkylphenyl, optionally
monosubstituted (C.sub.1-C.sub.4)alkoxyphenyl, or optionally
monosubstituted perhalo(C.sub.1-C.sub.4)alkylphenyl, wherein said
optional substitution is (C.sub.1-C.sub.4)alkyl, OH, or halogen;
R.sub.6 and R.sub.7 are independently H, alkyl, aryl, heteroaryl,
alkylaryl, arylalkyl, heteroarylalkyl, or alkylheteroaryl; provided
the compound is not
1-tert-butyl-3-p-tolyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamine.
In certain embodiments, Y is N.
In a detailed embodiment, R.sub.1a or R.sub.1b is phenyl
substituted with mono, di or tri-OH. In a further detailed
embodiment, the phenyl is further substituted with a halo. In a
further detailed embodiment, the halo is F.
In a detailed embodiment, R.sub.2 is 2-methyl-propane. In a
detailed embodiment, R.sub.3 and R.sub.4 are H. In a detailed
embodiment, R.sub.5 is H. In a detailed embodiment, R.sub.6 is H
and R.sub.7 is methyl.
In certain embodiments, R.sub.1a is, independently, phenyl
substituted at a meta position with --CH.sub.3, tert-butyl,
--CF.sub.3 or halo. In a detailed embodiment, R.sub.1a is,
independently, phenyl substituted at a meta position with halo,
alkyl, haloalkyl, haloaryl, aryl, O-alkyl, CN, NO.sub.2,
CO--O--R.sub.3, CO--N(R.sub.3).sub.2. In a detailed embodiment, Z
is F, Br Cl, or I
In a detailed embodiment, the compounds of formula I or formula II
include:
3-(4-amino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
[5-(3-amino-phenyl)-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-methyl-a-
mine;
3-(4-benzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol-
;
3-(4-dibenzylamino-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrinidin-7-yl]-propi-
onic acid tert-butyl ester;
3-[5-(3-hydroxy-phenyl)-4-methylamino-pyrrolo[2,3-d]pyrimidin-7-yl]-propi-
onic acid;
3-bromo-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-
-5-yl)-phenol;
3-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-ph-
enol;
3-tert-Butyl-5-(7-isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-
-5-yl)-phenol;
3-(7-Isopropyl-4-methylamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-trifluoro-
methyl-phenol;
3-bromo-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phenol;
3-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-methyl-phenol;
3-tert-butyl-5-(4-chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-phe-
nol;
3-(4-Chloro-7-isopropyl-7H-pyrrolo[2,3-d]pyrimidin-5-yl)-5-trifluorom-
ethyl-phenol or a pharmaceutically-acceptable salt or prodrug
thereof.
In a further detailed embodiment, the compound has the formula:
##STR00005##
In a further detailed embodiment, the compound has the formula:
##STR00006##
In a further detailed embodiment, the compound has the formula:
##STR00007##
In a further detailed embodiment, the compound has the formula:
##STR00008##
In a further detailed embodiment, the compound has the formula:
##STR00009##
In another embodiment, the pharmaceutical composition, comprises a
pharmaceutically acceptable carrier, and the compound. In a
detailed embodiment the pharmaceutical composition further
comprises at least one anthracycline compound, including but not
limited to, daunorubicin doxorubicin, epirubicin, idarubicin, or a
mixture thereof.
In another embodiment, methods for preventing or treating a disease
or condition associated with carbonyl reductase I in a mammalian
are provided, comprising the step of administering to the mammal a
composition comprising an effective amount of the compound.
In a further embodiment, the disease state is cancer. In a detailed
embodiment, the cancer is lung cancer.
In another embodiment, methods for identifying a therapeutic cancer
treatment are provided, comprising contacting a tumor cell culture
with an effective amount of the compound.
In another embodiment, methods for alleviating a disease state in a
mammal believed to be responsive to treatment with an inhibitor of
carbonyl reductase 1 are provided, comprising administering to the
mammal an effective amount of the compound, in combination with an
effective amount of an anthracycline anti-cancer agent, wherein the
disease state of the mammal is alleviated. In a detailed
embodiment, the anthracycline anti-cancer agent includes, but is
not limited to, daunorubicin, doxorubicin, epirubicin, or
idarubicin. In a further detailed embodiment, the potency of the
anthracycline anti-cancer agent is maintained in the absence of a
cardiotoxic side effect. In a detailed embodiment, the disease
state is cancer. In a further detailed embodiment, the disease
state is selected from cancer, metastatic cancer, colon cancer,
ovarian cancer, leukemia, lymphoma, myeloma, acute myeloid
leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,
neuroblastoma, lung cancer, breast cancer, acquired
immunodeficiency syndrome (AIDS)associated Kaposi's sarcoma
(KS),inflammation, obesity, or diabetes.
In another embodiment, methods of preventing or treating a disease
or condition associated with the synthesis of prostaglandin E in a
mammal comprises administering to the mammal a effective amount of
the compound wherein the disease state of the mammal is alleviated.
In a detailed embodiment, the disease state is metastatic cancer.
In a detailed embodiment, the disease state is colon cancer.
In another embodiment, methods for alleviating a disease state in a
mammal believed to be responsive to treatment with an inhibitor of
short chain dehydrogenase/reductase (SDR) family of NAD(P)(H)
dependent oxido-reductases, comprise administering to the mammal a
effective amount of the compound wherein the disease state of the
mammal is alleviated. In a detailed embodiment, the therapeutic
amount of the compound inhibits 11.beta.-hydroxysteroid
dehydrogenase I. In a detailed embodiment, the therapeutic amount
of the compound inhibits 11.beta.-hydroxysteroid dehydrogenase
II.
In a detailed embodiment, the therapeutic amount of the compound
stimulates synthesis of cortisol. In a further detailed embodiment,
the disease state is inflammation.
In a detailed embodiment, the therapeutic amount of the compound
stimulates degradation of cortisone. In a further detailed
embodiment, the therapeutic amount of the compound alleviates the
disease state selected from obesity or diabetes.
In a detailed embodiment, the therapeutic amount of the compound
inhibits 17.beta.-hydroxysteroid dehydrogenases. In a further
detailed embodiment, the therapeutic amount of the compound
alleviates the disease state selected from inflammation, ovarian
cancer or breast cancer.
In another embodiment, methods for identifying a therapeutic cancer
treatment are provided comprising the steps of: contacting a tumor
cell culture with an effective amount of a according to claim 1;
measuring growth inhibition of the tumor cells in culture; and
identifying a therapeutic cancer treatment for a mammalian subject
by inhibition of the tumor cell growth in culture
In another embodiment, methods for preventing or treating cancer in
a mammal are provided comprising the step of administering to the
mammal an effective amount of the compound. In a detailed
embodiment, the cancer is lung cancer, metastatic cancer, colon
cancer, ovarian cancer, leukemia, lymphoma, myeloma, acute myeloid
leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,
neuroblastoma, breast cancer, acquired immunodeficiency syndrome
(AIDS)-associated Kaposi's sarcoma (KS).
Pharmaceutical Compositions
Inhibitors and modulators of SDR type enzymes, for example,
AB129-type compounds and analogs thereof, are useful in the present
compositions and methods and can be administered to a human patient
per se, in the form of a stereoisomer, prodrug, pharmaceutically
acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide,
prodrug ester, or isomorphic crystalline form thereof, or in the
form of a pharmaceutical composition where the compound is mixed
with suitable carriers or excipient(s) in a therapeutically
effective amount, for example, lung cancer or colon cancer.
"Prodrug esters" as employed herein includes prodrug esters which
are known in the art for carboxylic and phosphorus acid esters such
as methyl, ethyl, benzyl and the like.
Routes of Administration
Pharmaceutical compositions of inhibitors and modulators of SDR
type enzymes, for example, AB129-type compounds and analogs
thereof, described herein can be administered by a variety of
routes. Suitable routes of administration can, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, spinal, epidural,
intranasal, or intraocular injections. Alternatively, one can
administer the compound in a local rather than systemic manner, for
example via injection of the compound directly into the subject,
often in a depot or sustained release formulation. Furthermore, one
can administer the compound in a targeted drug delivery system, for
example, in a liposome coated vesicle. The liposomes can be
targeted to and taken up selectively by the tissue of choice. In a
further embodiment, the pharmaceutical compositions of AB129-type
compounds and analogs described herein are administered orally.
Composition/Formulation
The pharmaceutical compositions described herein can be
manufactured in a manner that is itself known, e.g., by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Pharmaceutical compositions for use as described herein
can be formulated in conventional manner using one or more
physiologically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Proper
formulation is dependent upon the route of administration chosen.
For injection, the agents can be formulated in aqueous solutions,
e.g., in physiologically compatible buffers such as Hanks'
solution, Ringer's solution, or physiological saline buffer. For
transmucosal administration, penetrants appropriate to the barrier
to be permeated are used in the formulation. Such penetrants are
generally known in the art. For oral administration, the compounds
can be formulated readily by combining with pharmaceutically
acceptable carriers that are well known in the art. Such carriers
enable the compounds to be formulated as tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, suspensions and the
like, for oral ingestion by a patient to be treated. Pharmaceutical
preparations for oral use can be obtained by mixing the compounds
with a solid excipient, optionally grinding a resulting mixture,
and processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP). If desired, disintegrating
agents can be added, such as the cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate. Dragee cores are provided with suitable coatings. For
this purpose, concentrated sugar solutions can be used, which can
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments can be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds can
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers can be added. All formulations for oral administration
should be in dosages suitable for such administration. For buccal
administration, the compositions can take the form of tablets or
lozenges formulated in conventional manner. For administration by
inhalation, the compounds for use are conveniently delivered in the
form of an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g. gelatin for use in an inhaler or insufflator can
be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
The compounds can be formulated for parenteral administration by
injection, e.g., by bolus injection or continuous infusion.
Formulations for injection can be presented in unit dosage form,
e.g., in ampules or in multi-dose containers, with an added
preservative. The compositions can take such forms as suspensions,
solutions or emulsions in oily or aqueous vehicles, and can contain
formulatory agents such as suspending, stabilizing and/or
dispersing agents. Pharmaceutical formulations for parenteral
administration include aqueous solutions of the active compounds in
water-soluble form. Additionally, suspensions of the active
compounds can be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection
suspensions can contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension can also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Alternatively, the active ingredient can be in powder
form for constitution with a suitable vehicle, e.g., sterile
pyrogen-free water, before use.
The compounds can also be formulated in rectal compositions such as
suppositories or retention enemas, e.g., containing conventional
suppository bases such as cocoa butter or other glycerides. In
addition to the formulations described previously, the compounds
can also be formulated as a depot preparation. Such long acting
formulations can be administered by implantation (for example
subcutaneously or intramuscularly) or by intramuscular injection.
Thus, for example, the compounds can be formulated with suitable
polymeric or hydrophobic materials (for example as an emulsion in
an acceptable oil) or ion exchange resins, or as sparingly soluble
derivatives, for example, as a sparingly soluble salt.
A suitable pharmaceutical carrier for hydrophobic compounds is a
cosolvent system comprising benzyl alcohol, a nonpolar surfactant,
a water-miscible organic polymer, and an aqueous phase. The
cosolvent system can be the VPD co-solvent system. VPD is a
solution of 3% (w/v) benzyl alcohol, 8% (w/v) of the nonpolar
surfactant polysorbate 80, and 65% (w/v) polyethylene glycol 300,
made up to volume in absolute ethanol. The VPD co-solvent system
(VPD:5W) consists of VPD diluted 1:1 with a 5% (w/v) dextrose in
water solution. This co-solvent system dissolves hydrophobic
compounds well, and itself produces low toxicity upon systemic
administration. Naturally, the proportions of a co-solvent system
can be varied considerably without destroying its solubility and
toxicity characteristics. Furthermore, the identity of the
co-solvent components can be varied: for example, other
low-toxicity nonpolar surfactants can be used instead of
polysorbate 80; the fraction size of polyethylene glycol can be
varied; other biocompatible polymers can replace polyethylene
glycol, e.g. polyvinyl pyrrolidone; and other sugars or
polysaccharides can substitute for dextrose. Alternatively, other
delivery systems for hydrophobic pharmaceutical compounds can be
employed. Liposomes and emulsions are well known examples of
delivery vehicles or carriers for hydrophobic drugs. Certain
organic solvents such as dimethylsulfoxide also can be employed,
although usually at the cost of greater toxicity.
Additionally, the compounds can be delivered using a
sustained-release system, such as semipermeable matrices of solid
hydrophobic polymers containing the therapeutic agent. Various
types of sustained-release materials have been established and are
well known by those skilled in the art. Sustained-release capsules
can, depending on their chemical nature, release the compounds for
a few weeks up to over 100 days. The pharmaceutical compositions
also can comprise suitable solid or gel phase carriers or
excipients. Examples of such carriers or excipients include but are
not limited to calcium carbonate, calcium phosphate, various
sugars, starches, cellulose derivatives, gelatin, and polymers such
as polyethylene glycols.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions for administering the AB129 (see, e.g., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 18.sup.th
ed., 1990, incorporated herein by reference). The pharmaceutical
compositions generally comprise a differentially expressed protein,
agonist or antagonist in a form suitable for administration to a
patient. The pharmaceutical compositions are generally formulated
as sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
Effective Dosages
Pharmaceutical compositions suitable for use include compositions
wherein the AB129-type compounds and analogs are contained in a
therapeutically effective amount. Determination of an effective
amount is well within the capability of those skilled in the art,
especially in light of the detailed disclosure provided herein. For
any compound used in the present method, a therapeutically
effective dose can be estimated initially from cell culture assays.
For example, a dose can be formulated in animal models to achieve a
circulating concentration range that includes the IC.sub.50 as
determined in cell culture (i.e., the concentration of test
compound that is lethal to 50% of a cell culture) or the IC.sub.50
as determined in cell culture (i.e., the concentration of compound
that is lethal to 100% of a cell culture). Such information can be
used to more accurately determine useful doses in humans. Initial
dosages can also be formulated by comparing the effectiveness of
the AB129-type compounds and analogs described herein in cell
culture assays with the effectiveness of known cancer treatments.
In this method an initial dosage can be obtained by multiplying the
ratio of effective concentrations obtained in cell culture assay
for the AB129-type compounds and analogs and a known cancer
treatment by the effective dosage of the known cancer treatment.
For example, if an AB129-type compound or analog is twice as
effective in cell culture assay than the cancer treatment (i.e.,
the IC.sub.50 of AB129 is equal to one half times the IC.sub.50
cancer treatment in the same assay), an initial effective dosage of
the AB129-type compound or analog would be one-half the known
dosage for the cancer treatment. Using these initial guidelines one
having ordinary skill in the art could determine an effective
dosage in humans. Initial dosages can also be estimated from in
vivo data. One having ordinary skill in the art could readily
optimize administration to humans based on this data. Dosage amount
and interval can be adjusted individually to provide plasma levels
of the active compound which are sufficient to maintain therapeutic
effect. Usual patient dosages for oral administration range from
about 50-2000 mg/kg/day, typically from about 250-1000 mg/kg/day,
from about 500-700 mg/kg/day or from about 350-550 mg/kg/day.
Therapeutically effective serum levels will be achieved by
administering multiple doses each day. In cases of local
administration or selective uptake, the effective local
concentration of the drug can not be related to plasma
concentration. One having skill in the art will be able to optimize
therapeutically effective local dosages without undue
experimentation. The amount of composition administered will, of
course, be dependent on the subject being treated, on the subject's
weight, the severity of the affliction, the manner of
administration and the judgment of the prescribing physician. The
therapy can be repeated intermittently while lung cancer or colon
cancer is detectable or even when they are not detectable.
Moreover, due to its apparent nontoxicity, the therapy can be
provided alone or in combination with other drugs, such as for
example, anti-inflammatories, antibiotics, corticosteroids,
vitamins and the like. Possible synergism between the AB129-type
compounds or analogs described herein and other drugs can occur. In
addition, possible synergism between a plurality of AB129-type
compounds or analogs can occur.
The typical daily dose of a pharmaceutical composition of
inhibitors and modulators of SDR type enzymes, for example,
AB129-type compounds and analogs thereof, varies according to
individual needs, the condition to be treated and with the route of
administration. Suitable doses are in the general range of from
0.001 to 10 mg/kg bodyweight of the recipient per day. Within this
general dosage range, doses can be chosen at which the
pharmaceutical composition of AB129-type compounds and analogs has
a positive effect on cancer treatment efficacy. In general, but not
exclusively, such doses will be in the range of from 0.5 to 10
mg/kg.
In addition, within the general dose range, doses can be chosen at
which the compounds pharmaceutical composition of AB129-type
compounds and analogs has a positive effect on cancer treatment
efficacy. In general, but not exclusively, such doses will be in
the range of from 0.001 to 0.5 mg/kg. It is to be understood that
the 2 sub ranges noted above are not mutually exclusive and that
the particular activity encountered at a particular dose will
depend on the nature of the pharmaceutical composition of
AB129-type compounds and analogs used.
The pharmaceutical composition of AB129-type compounds and analogs
can be in unit dosage form, for example, a tablet or a capsule so
that the patient can self-administer a single dose. In general,
unit doses contain in the range of from 0.05-100 mg of a compound
of the pharmaceutical composition of AB129-type compounds and
analogs. Unit doses contain from 0.05 to 10 mg of the
pharmaceutical composition. The active ingredient can be
administered from 1 to 6 times a day. Thus daily doses are in
general in the range of from 0.05 to 600 mg per day. In an
embodiment, daily doses are in the range of from 0.05 to 100 mg per
day or from 0.05 to 5 mg per day.
Toxicity
Toxicity and therapeutic efficacy of inhibitors and modulators of
SDR type enzymes, for example, AB129-type compounds and analogs
thereof, described herein can be determined by standard
pharmaceutical procedures in cell cultures or experimental animals,
e.g., by determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index and can be expressed as
the ratio between LD.sub.50 and ED.sub.50 Compounds which exhibit
high therapeutic indices are chosen. The data obtained from these
cell culture assays and animal studies can be used in formulating a
dosage range that is not toxic for use in human. The dosage of such
compounds lies within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage can
vary within this range depending upon the dosage form employed and
the route of administration utilized. The exact formulation, route
of administration and dosage can be chosen by the individual
physician in view of the patient's condition. (See, e.g., Fingl et
al., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p.
1). One of the advantages, among others, of using the AB129-type
compounds and analogs described herein to treat disease, e.g., lung
cancer or colon cancer is their lack of toxicity. For example, it
has been found that repeated intraperitoneal doses of 75 mg/kg
produced no ill effects in mice (see Example 5). Since the i.v.
serum half-life (t.sub.1/2) of AB129 is about 2-2.5 hours, repeated
daily dosages of the AB129 described herein without ill effects is
predictable.
Diagnostic Methods
In addition to assays, the creation of animal models, and nucleic
acid based therapeutics, identification of important differentially
expressed genes allows the use of these genes in diagnosis (e.g.,
diagnosis of cell states and abnormal epithelial cell conditions).
Disorders based on mutant or variant differentially expressed genes
can be determined. Methods for identifying cells containing variant
differentially expressed genes comprising determining all or part
of the sequence of at least one endogenous differentially expressed
genes in a cell are provided. As will be appreciated by those in
the art, this can be done using any number of sequencing
techniques. Methods of identifying the differentially expressed
genotype of an individual comprising determining all or part of the
sequence of at least one differentially expressed gene of the
individual are also provided. This is generally done in at least
one tissue of the individual, and can include the evaluation of a
number of tissues or different samples of the same tissue. The
method can include comparing the sequence of the sequenced
differentially expressed gene to a known differentially expressed
gene, i.e., a wild-type gene.
The sequence of all or part of the differentially expressed gene
can then be compared to the sequence of a known differentially
expressed gene to determine if any differences exist. This can be
done using any number of known sequence identity programs, such as
Bestfit, and others outlined herein. In some methods, the presence
of a difference in the sequence between the differentially
expressed gene of the patient and the known differentially
expressed gene is indicative of a disease state or a propensity for
a disease state, as outlined herein.
Similarly, diagnosis of epithelial cell states can be done using
the methods and compositions herein. By evaluating the gene
expression profile of epithelial cells from a patient, the
epithelial cell state can be determined. This is particularly
useful to verify the action of a drug, for example an
immunosuppressive drug. Other methods comprise administering the
drug to a patient and removing a cell sample, particularly of
epithelial cells, from the patient. The gene expression profile of
the cell is then evaluated, as outlined herein, for example by
comparing it to the expression profile from an equivalent sample
from a healthy individual. In this manner, both the efficacy (i.e.,
whether the correct expression profile is being generated from the
drug) and the dose (is the dosage correct to result in the correct
expression profile) can be verified.
The present discovery relating to the role of differentially
expressed in epithelial cells thus provides methods for inducing or
maintaining differing epithelial cell states. In one method, the
differentially expressed proteins, and particularly differentially
expressed fragments, are useful in the study or treatment of
conditions which are mediated by epithelial cell activity, i.e., to
diagnose, treat or prevent epithelial cell-mediated disorders.
Thus, "epithelial cell mediated disorders" or "disease states" can
include conditions involving, for example, arthritis, diabetes, or
multiple sclerosis.
Methods of modulating epithelial cell activity in cells or
organisms are provided. Some methods comprise administering to a
cell an anti-differentially expressed antibody or other agent
identified herein or by the methods provided herein, that reduces
or eliminates the biological activity of the endogenous
differentially expressed protein. Alternatively, the methods
comprise administering to a cell or organism a recombinant nucleic
acid encoding a differentially expressed protein or modulator
including anti-sense nucleic acids. As will be appreciated by those
in the art, this can be accomplished in any number of ways. In some
methods, the activity of differentially expressed is increased by
increasing the amount of differentially expressed in the cell, for
example by overexpressing the endogeneous differentially expressed
or by administering a differentially expressed gene, using known
gene therapy techniques, for example. In one method, the gene
therapy techniques include the incorporation of the exogenous gene
using enhanced homologous recombination (EHR), for example as
described in PCT/US93/03868, hereby incorporated by reference in
its entirety.
Methods for diagnosing an epithelial cell activity related
condition in an individual are provided. The methods comprise
measuring the activity of differentially expressed protein in a
tissue from the individual or patient, which can include a
measurement of the amount or specific activity of the protein. This
activity is compared to the activity of differentially expressed
from either an unaffected second individual or from an unaffected
tissue from the first individual. When these activities are
different, the first individual can be at risk for an epithelial
cell activity mediated disorder.
Furthermore, nucleotide sequences encoding a differentially
expressed protein can also be used to construct hybridization
probes for mapping the gene which encodes that differentially
expressed protein and for the genetic analysis of individuals with
genetic disorders. The nucleotide sequences provided herein can be
mapped to a chromosome and specific regions of a chromosome using
known techniques, such as in situ hybridization, linkage analysis
against known chromosomal markers, and hybridization screening with
libraries.
Kits
The differentially expressed protein, agonist or antagonist or
their homologs are useful tools for examining expression and
regulation of signaling in epithelial cells via the PAR1 pathway.
Reagents that specifically hybridize to nucleic acids encoding
differentially expressed proteins (including probes and primers of
the differentially expressed proteins), and reagents that
specifically bind to the differentially expressed proteins, e.g.,
antibodies, are used to examine expression and regulation.
Nucleic acid assays for the presence of differentially expressed
proteins in a sample include numerous techniques are known to those
skilled in the art, such as Southern analysis, northern analysis,
dot blots, RNase protection, S1 analysis, amplification techniques
such as PCR and LCR, high density oligonucleotide array analysis,
and in situ hybridization. In in situ hybridization, for example,
the target nucleic acid is liberated from its cellular surroundings
in such as to be available for hybridization within the cell while
preserving the cellular morphology for subsequent interpretation
and analysis. The following articles provide an overview of the art
of in situ hybridization: Singer et al., Biotechniques 4: 230-250,
1986; Haase et al., Methods in Virology, vol. VII, pp. 189-226,
1984; and Nucleic Acid Hybridization: A Practical Approach (Hames
et al., eds. 1987), each incorporated herein by reference. In
addition, a differentially expressed protein can be detected with
the various immunoassay techniques described above. The test sample
is typically compared to both a positive control (e.g., a sample
expressing recombinant differentially expressed protein) and a
negative control.
Kits for screening epithelial cell activity modulators. Such kits
can be prepared from readily available materials and reagents are
provided. For example, such kits can comprise any one or more of
the following materials: the differentially expressed proteins,
agonists, or antagonists, reaction tubes, and instructions for
testing the activities of differentially expressed genes. A wide
variety of kits and components can be prepared depending upon the
intended user of the kit and the particular needs of the user. For
example, the kit can be tailored for in vitro or in vivo assays for
measuring the activity of a differentially expressed proteins or
epithelial cell activity modulators.
Kits comprising probe arrays as described above are provided.
Optional additional components of the kit include, for example,
other restriction enzymes, reverse-transcriptase or polymerase, the
substrate nucleoside triphosphates, means used to label (for
example, an avidin-enzyme conjugate and enzyme substrate and
chromogen if the label is biotin), and the appropriate buffers for
reverse transcription, PCR, or hybridization reactions.
Usually, the kits also contain instructions for carrying out the
methods.
Method of Preparation
The compounds of Formula I and II can be prepared in a number of
ways well known to those skilled in the art, including both solid
phase and solution techniques. The compounds can be synthesized,
for example, by the methods described below, or variations thereof
as appreciated by the skilled artisan. All processes disclosed in
association with the present invention are contemplated to be
practiced on any scale, including milligram, gram, multigram,
kilogram, multikilogram or commercial industrial scale.
As discussed in detail above, compounds of Formula I or II can
contain one or more asymmetrically substituted carbon atoms, and
can be isolated in optically active or racemic forms. Thus, all
chiral, diastereomeric, racemic forms and all geometric isomeric
forms of a structure are intended, unless the specific
stereochemistry or isomeric form is specifically indicated. It is
well known in the art how to prepare and isolate such optically
active forms. For example, mixtures of stereoisomers can be
separated by standard techniques including, but not limited to,
resolution of racemic forms, normal, reverse-phase, and chiral
chromatography, preferential salt formation, recrystallization, and
the like, or by chiral synthesis either from chiral starting
materials or by deliberate synthesis of target chiral centers.
As will be readily understood, functional groups present can
contain protecting groups during the course of synthesis.
Protecting groups are known per se as chemical functional groups
that can be selectively appended to and removed from
functionalities, such as hydroxyl groups and carboxy groups. These
groups are present in a chemical compound to render such
functionality inert to chemical reaction conditions to which the
compound is exposed. Any of a variety of protecting groups can be
employed with the present invention. Preferred protecting groups
include the benzyloxycarbonyl group and the tert-butyloxycarbonyl
group. Other preferred protecting groups that can be employed in
accordance with the present invention are described in Greene, T.
W. and Wuts, P. G. M., Protective Groups in Organic Synthesis 3d.
Ed., Wiley & Sons, 1991.
The compounds of Formula I, where Y is CR.sup.5, can be prepared as
shown in Scheme 1.
##STR00010##
In Scheme 1, the pyrrolopyrimidine scaffold
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine, compound 5, can
prepared from ethyl cyanoacetate and bromoacetaldehyde diethyl
acetal in six steps (10% yield). R.sup.2 is introduced by Mitsunobu
alkylation of compound 5, using either solid-phase or
solution-phase chemistry, to form compound 6. R.sup.2 substituents
can also be introduced by anion alkylation or Michael addition. A
4-formyl-3,5-dimethoxyphenoxymethyl-functionalized (PAL) resin is
loaded with an R.sup.4 appended amine by reductive amination to
form compound 7, employing, for example, methylamine, ethylamine,
benzylamine or 2,4,6-trimethoxybenzylamine or a suitable salt
thereof (such as the hydrogen chloride salt). Compounds 6 and 7 are
contacted and heated to allow the SNAR capture of the alkylated
scaffold to form compound 8. Using a solid-phase Suzuki coupling
employing the appropriate boronic acid and catalyst (such as
palladium), R.sub.1a is introduced to form compound 2.
Alternatively, a solution-phase Suzuki coupling can be employed.
Compound 2 is then cleaved from the solid support with
trifluoroacetic acid. Scheme 1 can be carried out under similar
reaction conditions as a solution-phase synthesis.
When a primary amine at R.sup.4 is required, a protecting strategy
can be employed. An acid-labile protecting group, such as, for
example, 2,4,6-trimethoxybenzylamine, is preferred. Acid labile
protecting groups for R.sup.1 or R.sup.2 substituents can also be
employed. Other suitable acid labile-protecting groups commonly
used in the art can be found in Greene and Wuts, Protective Groups
in Organic Synthesis, 2d ed, John Wiley & Sons, New York, 1991,
the disclosure of which is hereby incorporated by reference in its
entirety.
The compounds of Formula I and the compounds of Formula II of the
invention can be prepared as shown in Scheme 2. The compounds of
Formula I, where Y is N, can be prepared as generally described in
Hanefeld, U., Rees, C. W., White, A. J. P., Williams, D. J.,
"One-pot Synthesis of Tetrasubstituted Pyrazoles Proof of
Regiochemistry," J. Chem. Soc. Perkin Trans 1: 1545-1522, 1996, the
disclosure of which is incorporated herein by reference in its
entirety. Scheme 2 is also applicable where Z=halo or
Z=NR.sub.3R.sub.4, (wherein R.sub.3 and R.sub.4 are not hydrogen)
as demonstrated in Bishop, A. C. Chemical Genetic Approaches To
Highly Selective Protein Kinase Inhibitors, Ph. D. Doctoral
dissertation, Princeton University, 2000, the disclosure of which
is incorporated herein by reference in its entirety.
##STR00011##
##STR00012##
Solution phase synthesis of pyrrolopyrimidine derivatives is
carried out using the general scheme above. Reactions are analogous
to those employed during solid-phase synthesis. Mitsunobu
alkylation, anion alkylation or Michael addition type reactions can
be used to introduce R.sub.2 substituents in the production of 6.
SNaryl reaction of 6 with primary amines, secondary amines or
ammonia (R.sub.4.dbd.H), yields 9a. Suzuki coupling of an aryl
boronic acid or boronic ester yields 10. A subsequent deprotection
step is required when there are protecting groups as in the case
for RB11 synthesis.
##STR00013##
tert-Butyl
3-(4-chloro-5-iodo-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate:
The Michael addition was performed as follows:
4-Chloro-7,7a-dihydro-5-iodo-4aH-pyrrolo[2,3-d]pyrimidine (5. 3 g,
0.11 mol) and Cs.sub.2CO.sub.3 (5.25 g, 0.016 mol) were p within a
250 ml round bottom flask, and the contents were subject to high
vacuum for 20 min. The flask was purged with argon, t-Butylacrylate
(30 ml) was added and the reaction was left to stir at room
temperature overnight. The reaction was quenched with 100 ml 10%
aqueous mono-sodium citrate, and the organic materials were
extracted into ethyl acetate (3.times.100 ml). The combined
organics were dried with sodium sulfate, and evaporated in vacuo to
yield a viscous oil. Silica gel chromatography (ethyl
acetate:hexanes) and evaporation of the requisite fractions yielded
0.7 g (17.2% yield) of the desired product as a white solid.
.sup.1H NMR (399.6 MHz, CDCl.sub.3) .delta. 1.38 (9H, s), 2.76 (2H,
t, J=6.4 Hz), 4.50 (2H, t, J=6.4 Hz), 7.47 (1H, s), 8.59 (1H,
s).
tert-butyl
3-(5-iodo-4-(methylamino)-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)
propanoate: t-Butyl
3-(4-chloro-5-iodo-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoate
from above (0.05 g, 0.12 mmol) was placed in a 15 ml pressure tube.
2M methylamine in THF (7 ml) was added and the vessel was sealed
and left to stir overnight. The volatiles were evaporated in vacuo,
the resultant material was quenched with 20 ml 10% monosodium
citrate, and the solution was extracted with ethyl acetate
(3.times.20 ml). The combined organic extracts were dried with
sodium sulfate and evaporated in vacuo. The resultant product
(0.069 g, 140% yield) was used without further purification.
.sup.1H NMR (399.6 MHz, CDCl.sub.3) .delta. 1.38 (s), 2.71 (2H, t,
J=6.4 Hz), 3.15 (3H, d, J=4.8 Hz), 4.38 (2H, t, J=6.4 Hz), 6.04
(3H, app s), 7.04 (s), 8.33 (s).
tert-Butyl
3-(5-(3-hydroxyphenyl)-4-(methylamino)-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-
-yl)propanoate (RB10): t-butyl
3-(5-iodo-4-(methylamino)-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-yl)propanoat-
e (123 mmol) from above was placed in a 25 ml round bottom flask,
whereupon 3.1 ml dimethoxy ethyleneglycol was added.
3-Hydroxyphenylboronic acid (492 mmol pre-dissolved in 0.66 ml
ethanol) was added at once, and was followed by 0.5 ml saturated
aqueous sodium carbonate. Pd.sup.0(PPh.sub.3).sub.4 (14 mg, 12
umol) was added to the reaction, the vessel was purged with argon,
and set to stir at 80 C overnight. The reaction was subsequently
cooled, and filtered through a bed of celite. The filtrate was
evaporated in vacuo, and the residual material was adhered to
silica gel using ethyl acetate as solvent. Silica gel
chromatography (ethyl acetate:hexanes) and evaporation in vacuo of
the requisite fractions yielded the desired product. MS
m/z=369.22.
3-(5-(3-hydroxyphenyl)-4-(methylamino)-4aH-pyrrolo[2,3-d]pyrimidin-7(7aH)-
-yl)propanoic acid (RB11): RB10 (9.6 mg, 26 umol) was treated with
2 ml deprotection solution (45% TFA, 45% CH.sub.2Cl.sub.2, 5%
Me.sub.2S, 5% H.sub.2O) for 1 hr at room temperature. The volatiles
were evaporated in vacuo, and 1 ml acetonitrile:water:TFA
(1:1:0.002) was added. The resultant solution was purified by
reverse-phase HPLC using a linear gradient of water to acetonitrile
both containing 0.1% TFA. The requisite fractions were pooled and
lyophilized to give the desired product 4.1 mg (79% yield) as a
white powder. .sup.1H NMR (399.6 MHz, d.sup.6-DMSO) .delta. 2.85
(2H, t, J=6.4 Hz), 3.0 (3H, d, J=4.4 Hz), 4.3, t, J=6.4 Hz), 6.8
(3H, m), 7.27 (1H, app t, J=7.6 Hz), 9.55 (1H, b s) The amino
proton resonance was presumably hidden due to the presence of water
in the NMR sample.
##STR00014##
The general solution phase synthetic strategy was used to
synthesize RB6 in 3 steps from compound 5. RB8 and RB9 were
produced in a similar manner, except benzyamine and dibenzyl amine
respectively were used during the S.sub.Naryl reaction.
Mitsunobu alkylation of 5 with isopropanol: To a dry 50 ml round
bottom flask was added 5 (0.5 g, 1.78 mmol) and PPh.sub.3 (0.84 g,
3.2 mmol). The materials were dried under high vacuum for 20 m, and
the flask was purged with argon. THF (30 ml) and isopropanol (0.3
ml, 3.9 mmol) were added and the flask was cooled in an ethylene
glycol/dry ice bath whereupon DiAD (0.47 g, 2.3 mmol) was added
dropwise to the stirred solution. After 18 h, the volatiles were
evaporated in vacuo and the resultant oil was dissolved in ethyl
acetate (50 ml) and 50% saturated sodium bicarbonate (50 ml). The
organics were extracted with ethyl acetate (3.times.50 ml), dried
with sodium sulfate and evaporated in vacuo to yield an orange oil.
Silica gel chromatography (ethyl acetate:hexanes) afforded the
desired product as a yellow solid (480 mg, 84% yield). .sup.1H NMR
(399.6 MHz, CDCl.sub.3) .delta. 1.5 (6H, d, J=6.4 Hz), 5.1 (1H, sp,
J=6.8 Hz), 7.4 (1H, s), 8.6 (1H, s).
7,7a-Dihydro-5-iodo-7-isopropyl-N-methyl-4aH-pyrrolo[2,3-d]pyrimidin-4-am-
ine:
4-Chloro-7,7a-dihydro-5-iodo-7-isopropyl-4aH-pyrrolo[2,3-d]pyrimidine
(0.3 g, 0.93 mmol) from above was placed within a 15 ml pressure
tube. 2 M methylamine in THF (15 ml) was added, and the reaction
was left to stir overnight. The volatiles were removed in vacuo,
and the residue was dissolved in methanol, 5 ml silica gel were
added, and the volatiles were removed in vacuo. The adhered product
was purified by silica gel chromatography (ethyl acetate:hexanes),
and the requisite fractions were pooled and evaporated in vacuo to
yield the desired product (0.25 g, 85% yield). .sup.1H NMR (399.6
MHz, CDCl.sub.3) .delta. 1.43 (6H, d, J=6.8 Hz), 3.13 (3H, d, J=4.8
Hz), 5.0 (1H, sp, J=6.8 Hz), 7.02 (1H, s), 8.35 (1H, s).
7,7a-Dihydro-7-isopropyl-N-methyl-5-phenyl-4aH-pyrrolo[2,3-d]pyrimidin-4--
amine (RB6):
7,7a-Dihydro-5-iodo-7-isopropyl-N-methyl-4aH-pyrrolo[2,3-d]pyrimidin-4-am-
ine (0.15 g, 0.475 mmol) from above was placed in a 50 ml round
bottom flask, whereupon 12 ml dimethoxy ethyleneglycol was added.
3-Hydroxyphenylboronic acid (0.262 g, 1.9 mmol pre-dissolved in 3.3
ml ethanol) was added at once, and was followed by 1.9 ml saturated
aqueous sodium carbonate. Pd.sup.0(PPh.sub.3).sub.4 (55 mg, 47
umol) was added to the reaction, the vessel was purged with argon,
and set to stir at 80 C for 48 h. The reaction was subsequently
cooled, and filtered through a bed of celite. The filtrate was
evaporated in vacuo, and the residual material was adhered to
silica gel using ethyl acetate as solvent. Silica gel
chromatography (ethyl acetate:hexanes) and evaporation in vacuo of
the requisite fractions yielded the desired product (94.8 mg, 70.7%
yield). .sup.1H NMR (399.6 MHz, d.sup.6-DMSO) .delta. 1.76 (6H, d,
J=6.8 Hz), 5.03 (3H, d, J=4.8 Hz), 5.34 (1H, sp, J=6.4 Hz), 5.53
(1H, q, J=4.8 Hz), 6.73 (1H, m), 6.85 (1H, m), 7.25 (1H, app t,
J=7.6 Hz), 7.37 (1H, s), 7.59 (1H, s).
Anion alkylation of 5 with methyl iodide: To a dry 50 ml round
bottom flask was added 5 (0.2 g, 0.7 mmol) and 15 ml dry
acetonitrile. The reaction was cooled on ice, and NaH (0.026 g, 1.1
mmol) was added at once. After stirring for 5 m, MeI (0.152 g, 1.07
mmol) was added dropwise. The reaction was allowed to warm to room
temperature overnight. The volatiles were evaporated, the residue
was dissolved in ethyl acetate:water, and the organics were
extracted with ethyl acetate (3.times.50 ml). The organics were
dried with sodium sulfate and evaporated in vacuo. The residue was
subject to silica gel chromatography (ethyl acetate:hexanes). The
requisite fractions were pooled and evaporated to yield a granular
solid (0.12 g, 56% yield). .sup.1H NMR (399.6 MHz, CDCl.sub.3)
.delta. 3.87 (1H, s), 7.35 (1H, s), 8.62 (1H, s).
##STR00015##
##STR00016##
3-Substituted anisoles were either purchased from Aldrich
(X=CH.sub.3, CF.sub.3, Br) or synthesized from the corresponding
phenols (X=isopropyl, t-butyl). Direct borylation was performed
according to the general procedures described by Miyaura and
Hartwig. Ishiyama, T.; et al., J. Am. Chem. Soc., 124: 390-391,
2002; Ishiyama, T.; et al., Angew. Chem. Int. Ed., 41: 3056-3058,
2002. A typical experimental procedure is given below.
3-Trifluoromethyl-
5-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)anisole. A
flame-dried 100 mL Schlenk tube was charged with
bis(pinacolato)diboron (350 mg, 1.38 mmol), [Ir(COD)Cl].sub.2 (12
mg, 0.018 mmol), sodium methoxide ( 5 mg, 0.09 mmol), and
4,4'-di-tert-butyl-2,2'-dipyridyl (8 mg, 0.03 mmol). The flask was
evacuated, placed under argon, and 3-trifluoromethylanisole (2.5
mL) was added. The flask was restoppered and evacuated (full
vacuum, 2 minutes). The flask was sealed under vacuum and
maintained at 90.degree. C. (oil bath) for 96 h. Thereafter, the
contents were transferred to a round bottom flask with the aid of
ethyl acetate and purified by Kugelrohr distillation. The product,
a viscous oil, distills at 120.degree. C. @10 .mu.m. Isolated yield
497 mg (1.65 mmol, 60%). .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.
7.62 (s, 1 H); 7.43 (s, 1 H); 7.18 (s, 1 H); 3.82 (s, 1 H); 1.32
(s, 1 H).
##STR00017##
The appropriate aryl pinacol borate (0.1 mmol) and iodinated
substrate (0.1 mmol) were dissolved in acetone and transferred to a
Schlenk flask. The solvent was evaporated and the flask was charged
with Pd(PPh.sub.3).sub.4 (3 mg) and K.sub.3PO.sub.4 (100 mg). The
flask was evacuated, placed under argon, and charged with 5 mL of
degassed anhydrous DMF. The resulting solution was heated at
60.degree. C. for 24 h under argon. Water was added and the mixture
was extracted (3.times.10 mL) with ethyl acetate. The combined
organic fractions were washed with water and saturated aqueous
NaCl, dried over Na.sub.2SO.sub.4, and evaporated. The remaining
material was loaded unto a small (0.5 cm.times.8 cm) silica gel
column and eluted with 1:4 ethyl acetate:hexane solution. Isolated
yields ranged from 50% to 90%.
X=CH.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.62 (s, 1 H);
7.32 (s, 1 H); 6.91 (s, 1 H); 6.87 (s, 1 H); 6.73 (s, 1 H); 5.18
(septet, 1 H, J=6.8 Hz); 3.82 (s, 3 H); 2.37 (s, 3 H); 1.54 (d, 6
H, J=6.8 Hz).
X=CF.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.64 (s, 1 H);
7.37 (s, 1 H); 7.34 (s, 1 H); 7.23 (s, 1 H); 7.11 (s, 1 H); 5.19
(septet, 1 H, J=6.7 Hz); 3.88 (s, 3 H); 1.56 (d, 6 H J=6.7 Hz).
X=Br .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.63 (s, 1 H); 7.33
(s, 1 H); 7.23 (s, 1 H); 7.04 (s, 1 H); 6.99 (s, 1 H); 5.18
(septet, 1 H, J=6.8 Hz); 3.83 (s, 3 H); 1.55 (d, 6 H, J=6.8
Hz);
X=tert-butyl .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.61 (s, 1
H); 7.33 (s, 1 H); 7.15 (s, 1H); 6.91 (s, 1 H); 6.82 (s, 1 H); 5.18
(septet, 1 H, J=6.8 Hz); 3.81 (s, 3 H); 1.56 (d, 6 H, J=6.8 Hz);
1.35 (s, 9 H).
X=CO.sub.2CH.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.63
(s, 1 H); 7.76 (t, 1 H, J=1.4 Hz); 7.54 (dd, 1 H, J.sub.1=2.4 Hz,
J.sub.1=1.4 Hz); 7.36 (s, 1 H); 7.26 (dd, 1 H, J.sub.1=2.4 Hz,
J.sub.1=1.4 Hz); 5.18 (septet, 1 H, J=6.8 Hz); 3.91 (s, 3 H); 3.88
(s, 3 H); 1.55 (d, 6 H, J=6.8 Hz).
##STR00018##
General Procedure: The anisole derivative (20 mg) was dissolved in
methylene chloride (5 mL) and transferred to an argon flushed
Schlenk tube. The solution was chilled to 0.degree. C. before 1 mL
of a BBr3 solution (1 M in CH2Cl2) was added. The mixture was
stirred at 0.degree. C. for 2 h. Saturated aqueous NaHCO.sub.3 was
added, the biphasic mixture was stirred for 15 min, extracted with
CH.sub.2Cl.sub.2, and the organic extracts were dried over Na2SO4.
The solvent was evaporated and the organic residue was purified by
flash chromatography on silica gel (1:1 hexane:ethyl acetate
eluant). Isolated yields were in excess of 80%.
X=CH3; 1H NMR (400 MHz, CDCl3) .delta. 8.62 (s, 1 H); 7.31 (s, 1
H); 6.87 (s, 1 H); 6.81 (s, 1 H); 6.69 (s, 1 H); 5.77 (broad
singlet, 1 H); 5.17 (septet, 1 H, J=6.7 Hz); 2.34 (s, 3 H); 1.54
(d, 6 H, J=6.7 Hz).
X=CF3; 1H NMR (400 MHz, CDCl3) .delta. 8.66 (s, 1 H); 7.40 (s, 1
H); 7.28 (s, 1 H); 7.20 (s, 1 H); 7.11 (s, 1 H); 5.19 (septet, 1 H,
J=6.8 Hz); 1.56 (d, 6 H J=6.8 Hz).
X=Br; 1H NMR (400 MHz, CDCl3) .delta. 8.32 (s, 1 H); 7.09 (s, 1 H);
7.09 (s, 1 H); 7.00 (s, 1 H); 6.65 (s, 1 H); 5.27 (broad quart., 1
H, J=4.6); 5.03 (septet, 1 H, J=6.8 Hz); 3.12 (d, 3 H, J=4.6 Hz);
1.48 (d, 6 H, J=6.8 Hz).
X=tert-butyl; 1H NMR (400 MHz, CDCl3) .delta. 8.64 (s, 1 H); 7.35
(s, 1 H); 7.08 (s, 1 H); 6.88 (s, 1 H); 6.80 (s, 1 H); 5.19
(septet, 1 H, J=6.7 Hz); 3.19 (d, 3 H, J=4.6 Hz) 1.52 (d, 6 H,
J=6.7 Hz); 1.34 (s, 9 H).
##STR00019##
General Procedure: Each compound (20 mg) was dissolved in 5 mL of a
THF solution containing methyl amine (1 M) and transferred to an
argon flushed 50 mL Schlenk storage tube. The vessel was sealed and
heated at 60.degree. C. for 24 h. The THF was evaporated and the
remainder was partitioned between ethyl acetate and aqueous
bicarbonate solution. The biphasic mixture was extracted with ethyl
acetate and the combined organic extracts were dried over
Na.sub.2SO.sub.4. The solvent was evaporated and the organic
residue was purified by flash chromatography on silica gel (1:3
hexane:ethyl acetate eluant). Isolated yields were in excess of
80%.
X=CH.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.38 (s, 1 H);
7.02 (s, 1 H); 6.75 (s, 1 H); 6.75 (s, 1 H); 6.71 (s, 1 H); 5.67
(broad s, 1 H); 5.07 (septet, 1 H, J=6.7 Hz); 3.16 (broad d, 3 H,
J=4.7 Hz); 2.34 (s, 3 H) 1.48 (d, 6 H, J=6.7 Hz).
X=CF.sub.3 .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.38 (s, 1 H);
7.19 (s, 1 H); 7.15 (s, 1 H); 7.10 (s, 1 H); 7.10 (s, 1 H); 5.50
(broad s, 1 H); 5.09 (septet, 1 H, J=6.7 Hz); 3.20 (d, 3 H, 4.9 Hz)
1.52 (d, 6 H J=6.7 Hz).
X=Br .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.64 (s, 1 H); 7.35
(s, 1 H); 7.19 (t, 1 H, J=1.5 Hz); 7.04 (t, 1 H, J=2.0 Hz); 6.95
(dd, 1 H, J.sub.1=2.0 Hz, J.sub.2=1.5 Hz); 5.17 (septet, 1 H, J=6.8
Hz); 1.54 (d, 6 H, J=6.8 Hz).
X=t-butyl .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 8.42 (s, 1 H);
7.06 (s, 1 H); 6.96 (s, 1 H); 6.94 (s, 1 H); 6.82 (s, 1 H); 5.25
(broad s, 1 H) 5.10 (septet, 1 H, J=6.7 Hz); 3.81 (s, 3 H); 1.55
(d, 6 H, J=6.8Hz); 1.32 (s, 9 H).
Other embodiments and uses will be apparent to one skilled in the
art in light of the present disclosures.
Exemplary Embodiments
Development of potent and selective inhibitors of individual SDR
family members have the potential to increase the local
concentration of endogenous hormones with important therapeutic
benefits such as cortisol as an anti-inflammatory agent
(11.beta.-hydroxysteroid dehydrogenase II), or to block production
of potent chemoattractants such as prostaglandin E2 for blocking
colon cancer or metastatic cancer (Carbonyl reductase 1; FIG. 1)
(Forrest, G. L. et al., Chem Biol Interact, 129: 21-40, 2000), or
for degradation of agents that cause obesity or glucose intolerance
leading to insulin resistant diabetes such as cortisone (
11.beta.-hydroxysteroid dehydrogenase I; FIG. 2) (Oppermann, U. C.,
et al., Chem Biol Interact, 130-132(1-3): 699-705, 2001). Of
particular focus here is the potential for blocking the action of
Carbonyl Reductase 1, which is responsible for the reduction of the
C-13 keto group of the anthracycline anti-cancer agents
(daunorubicin: Cerubidin.RTM., DaunoXome.RTM.; doxorubicin:
Adriamycin.RTM.) (FIG. 3). The reduction of C-13 keto of
adriamycin, inactivates the anti-cancer activity of daunorubicin
and produces a product (daunorubicinol) which is known to be
cardiotoxic. Thus, blocking the action of CBR1 in patients treated
with adriamycin, would be predicted to enhance the potency of
adriamycin's anti-cancer activity and also to reduce the harmful
cardiotoxic effects of the adriamycin metabolite (daunorubicinol).
Thus, the SDR family members represent an important class of
enzymes critical for control of the biological activity of a wide
variety of endogenous and xenobiotics compounds. By designing
inhibitors of individual members of this family of enzymes new
therapies for lung cancer, breast cancer, obesity, diabetes, and
for improving the activity and decreasing the toxicity of existing
anti-cancer drugs should be possible.
Microarray studies (67 tumors from 56 patients) show that CBR1 is
upregulated in squamous cell lung carcinoma, but not in small cell
lung carcinoma, (FIG. 4; M. E. Garber et al., Proc. Nat. Acad. Sci
USA, 98: 13784, 2001) leading to the hypothesis that AB129 kills
such cells by inhibiting CBR1.
Inhibitory activity of AB129 has been demonstrated by measuring
reduction of menadione to mendadiol by carbonyl reductase 1 (CBR1).
AB129 inhibits the CBR1 catalyzed reduction of menadione by NADPH.
The IC.sub.50 for AB129 was approximately 5 .mu.M. PP1 did not
inhibit CBR1. (FIG. 5) At a concentration as high as 16 .mu.M.
AB129 is a competitive inhibitor of CBR1, with respect to NADPH
(FIG. 6).
Experiments using interfering RNA (RNAi) downregulate CBR1 by
inhibiting translation of mRNA in A549 lung carcinoma cells and
demonstrate that CBR1 has a role in development of lung cancer.
RNAi inhibition of CBR1 translation demonstrates a 60 to 70%
decrease in viability of A549 lung carcinoma cells compared to an
approximately 50% decrease in viability of A549 cells in the
presence of AB129. (FIGS. 7 and 8) This suggests that inhibition of
CBR1 expression in A549 cells decreases cell viability.
EXAMPLE 1
The SDR Family Member 11.beta.-Hydroxysteroid Dehydrogenase 2
(11.beta.-HSD2) Controls the Local Metabolism of Glucocorticoids
and Directly Regulates Tissue Specific Nuclear Hormone
Signaling
Classical small molecule ligand/receptor pairs in biology interact
when both are present in the same tissue and are structurally
complementary to one another. An important exception to this
paradigm is that of the mineralcorticoid receptor which binds both
cortisol and aldosterone (Funder, J. W., et al., Science, 242:
583-5, 1988). In the kidney, the mineralcorticoid receptor
regulates K.sup.+ uptake and water absorption, in response to the
rennin-angiotensin-aldosterone signaling cascade. However, since
blood concentrations of cortisol are 100-1000 fold greater than
aldosterone, the mineralcorticoid receptor must be "protected" from
activation by cortisol in order to allow proper regulation by
aldosterone. This "protective" function is carried out by
11.beta.-HSD2 co-localized with the mineralcorticoid receptor,
which converts cortisol to cortisone, a steroid hormone which has
no binding affinity for the mineralcorticoid receptor (FIG. 2)
(Funder, J. W., et al., Science, 242: 583-5, 1988). Congenital loss
of this enzyme causes apparent mineralcorticoid receptor excess,
due to overstimulation of the mineralcorticoid receptor by
cortisol, bypassing its normal regulation by aldosterone (White, P.
C., et al., Endocr Rev, 18: 135-56, 1997). This unusual mechanism
of nuclear receptor ligand regulation suggests a potentially new
therapeutic approach to treat asthma.
EXAMPLE 2
Targeting 11.beta.-Hydroxysteroid Dehydrogenase 2 (11.beta.-HSD2)
as a Potential Alternative to Synthetic Corticosteroid Treatment of
Asthma
Tissue specific metabolism of steroids is an important factor in
regulating the properties of endogenous steroids, perhaps drugs
which inhibit these enzymes could effectively regulate the local
concentrations of beneficial endogenous hormones. In asthma, local
administration of an inhibitor of 11.beta.-HSD2 in the lung would
block conversion of the anti-inflammatory steroid, cortisol to
inactive cortisone, thus providing a larger concentration of the
body's own anti-inflammatory agent to reduce bronchial swelling in
this tissue. One potential benefit is that cortisol in the lung
would remain regulatable by tissue specific metabolizing enzymes
outside of the lung. Synthetic glucocorticoids currently used in
asthma therapy are not metabolized by these enzymes, and thus can
show pleiotropic effects in other tissues if dosages are not
controlled (Barnes, P. J. et al., Am Rev Respir Dis, 148: S1-26,
1993). Thus, the strategy proposed here, can avoid the
complications of administration of synthetic corticosteroids to
patients which can cause high blood pressure, swelling, changes of
mood and weight gain, all of which are known functions of cortisol
in the body, but which are unwanted side-effects for asthma
patients.
The strategy of regulating the metabolism of cortisol through
11.beta.-HSD2 inhibition in the lung is less prone to unwanted
side-effects than synthetic glucocorticoid therapy, even though
both act through glucocorticoid receptors which are present
throughout the body. Vastly different levels of 11.beta.-HSD2 are
expressed in the lung compared to other organs, providing an avenue
for potent inhibition in the lung without significant inhibition
systemically. 11.beta.-HSD2 is expressed at significantly lower
levels in the lung compared to the kidney, adrenal and colon
(Romero, D. G., et al., J Steroid Biochem Mol Biol, 72: 231-7,
2000). One caveat is that only the mRNA levels have been reported
which may not directly correspond to a difference in 11.beta.-HSD2
protein levels. By administering a relatively low dose of an
11.beta.-HSD2 inhibitor directly in the lung (intratracheal in the
mouse, or with a nebulizer in patients), a significant inhibition
of 11.beta.-HSD2 is achieved in the lung, resulting in a
significant increase of cortisol concentration. Any of the
inhibitor which is absorbed systemically will encounter much larger
concentrations of 11.beta.-HSD2, and thus will be unable to
significantly perturb 11.beta.-HSD2 function in these tissues,
resulting in less severe side-effects compared to synthetic
glucocorticoid therapy. Thus, the different levels of 11.beta.-HSD2
expression in the lung can provide additional control over unwanted
systemic glucocorticoid stimulation in asthma patients.
EXAMPLE 3
Evidence Linking 11.beta.-Hydroxysteroid Dehydrogenase 2
(11.beta.-HSD2) Inhibition as a Therapy for Asthma
Schleimer and coworkers have suggested 11.beta.-HSD2 is an
attractive target for treatment of asthma (Feinstein, M. B. et al.,
Am J Respir Cell Mol Biol, 21: 403-8, 1999). They point out that a
natural product isolated from licorice is an ancient therapy for
asthma and many other inflammatory diseases such as eczema and
Addison's disease (Persson, C. G., Pulm Pharmacol, 2: 163-6, 1989).
The major bioactive component of licorice, glycyrrhizic acid is in
fact an inhibitor of 11.beta.-HSD2 (IC.sub.50=8 nM) (FIG. 9)
(Diederich, S., e al., Eur J Endocrinol, 142: 200-7, 2000).
Schleimer and coworkers first confirmed that 11.beta.-HSD2 is
present in lung epithelial cells, and that cortisol is rapidly
oxidized to cortisone in this tissue. Next, they confirmed that
glycyrrhizic acid's anti-inflammatory activity in cells is
dependent on the presence of 11.beta.-HSD2, further supporting the
link between this natural product and 11.beta.-HSD2. Unfortunately,
glycyrrhizic acid is not likely to be a very good asthma therapy
because of its non-selective nature. It inhibits 11.beta.-HSD1,
which blocks production of cortisol from cortisone, resulting in a
reduction of anti-inflammatory cortisol concentration, at almost
equivalent potency to its inhibition of 11.beta.-HSD1 (IC.sub.50=40
nM) (Diederich, S., e al., Eur J Endocrinol, 142: 200-7, 2000). In
fact, recent studies with a derivative of glycyrrhizic acid,
carbenoxolone, with similar potency and specificity for
11.beta.-HSD1 and 2 (FIG. 2), has shown potent inhibition of
11.beta.-HSD2 in men with type 2 diabetes (11.beta.-HSD2 inhibition
was not measured) (Andrews, R. C., et al., J Clin Endocrinol Metab,
88: 285-91, 2003).
Glycyrrhizic acid's non-selective nature is due to its interaction
with the glucocorticoid binding pocket of 11.beta.-HSD 1 & 2
which is conserved between the two enzymes. In fact, both enzymes
can operate as a reductase or an oxidase, catalyzing both formation
and degradation of cortisol. In the body, the directionality of
each enzyme is controlled by regulation of re-dox cofactor
concentration with NADPH preferred by 11.beta.-HSD1 and NAD.sup.+
preferred by 11.beta.-HSD2 (Diederich, S., e al., Eur J Endocrinol,
142: 200-7, 2000). A highly selective 11.beta.-HSD2 inhibitor is
developed by targeting the NAD.sup.+ binding pocket of
11.beta.-HSD2 which is differentiated from that of 11.beta.-HSD 1
by the preference of 11.beta.-HSD2 for NAD.sup.+ as a cofactor and
preference of 11.beta.-HSD 1 for NADPH. In fact, a similar approach
has been successfully used to design selective inhibitors of
11.beta.-HSD 1, which is an attractive drug target for treatment of
obesity and insulin resistant diabetes (Barf, T., et al., J Med
Chem, 45: 3813-5, 2002). The same strategy is applied for treatment
of asthma by targeting 11.beta.-HSD2.
EXAMPLE 4
Discovery of an Inhibitor of the SDR Family Member, Carbonyl
Reductase 1 (CBR1)
Three structurally similar compounds (AB129, 1, AB60, 3, and AB61,
4), but not PP1, 2 (FIG. 10) were found to cause mild to severe
cell killing in the human lung cancer cell line, A549. (FIG. 11)
AB129 affects the cell cycle in A549 cells with approximately 12%
of A549 cells in G2/M phase, whereas PP1-treated A549 cells have
approximately 5.6% of cells in G2/M phase. AB129 treated cells show
a proportion of cells that may be polyploid. (FIG. 12). Many small
molecules with cell killing activity on cancer cell lines have been
described to date (REFs) yet often the targets of the small
molecules cannot be identified because the compounds bind poorly
(>1 .mu.M Kd) to the targets, or the targets are expressed at
very low abundance (<100,000 copies/cell), or derivatization of
the small molecule necessary for attachment of an affinity tag
(biotin) or attachment to a bead (for affinity purification of the
target protein) reduces the cellular activity and thus ability to
bind the target. A strategy was pursued to identify the target or
targets of the AB129, AB60, and AB61 compounds based on affinity
chromatography.
A derivatized form of AB129 was synthesized. The AB129 compound
produced the most potent A549 cell killing response. The
derivatized compound was bound to an agarose bead (P in FIG. 13)
and cell lysates were passed over the beads, hoping to retain the
true target of AB129 and eliminate all non-interacting proteins.
Importantly, a control resin (C in FIG. 13) was used to determine
if any interacting proteins were truly specific for AB129, or were
common binders of the pyrazolopyrimidine scaffold. The results of
the affinity purification (pull-down) experiment are shown in FIG.
14. This type of approach is successful in cases when the target
affinity is high (<1 .mu.M) and when the site of attachment to
the bead does not perturb the binding to the cellular target
(Mayer, T. U., et al., Science, 286: 9714, 1999; Kwok, B. H., et
al., Chem Biol, 8: 759-66, 2001). Typically, hits from forward
chemical genetic screens are of poor potency (>20 .mu.M), and
thus the affinity capture strategy is unsuccessful.
Using mass spectrometry the proteins retained on the AB129 beads
were analyzed and three proteins identified, including carbonyl
reductase 1 (CBR1) (FIGS. 15-22). To confirm that CBR1 is inhibited
by AB129 CBR1 was expressed in bacteria. It was shown that AB129 is
a pure NADPH competitive inhibitor of CBR1, with a Ki of 400 nM
(FIG. 23). This is a very potent compound for an initial hit in a
broad based screen. Importantly, the known kinase inhibitor, PP1,
(FIG. 5) does not inhibit CBR1, in this in vitro assay, nor does
CBR1 bind to control PP1-agarose beads, used as a control for
affinity purification of the targets of AB129 (FIG. 14).
To understand the basis for the potency of AB129 against CBR1, a
computer algorithm was used for molecular docking of AB129 to an
available crystal structure of porcine CBR1 to produce a model of
the bound structure of AB129 (FIG. 24). This modeled co-structure
was experimentally validated by site-directed mutagenesis of
multiple amino acids in the proposed AB129 binding pocket
(including a conserved Asn residue common to all SDR family
members--FIGS. 25-27) and identification of AB129 resistant mutants
of CBR1 (FIGS. 28-30). This binding model also potentially explains
the importance of the hydroxyl moiety attached to the phenyl ring
of AB129, as being critical for a H-bonding interaction with an
active site Asn. Since PP1 lacks this key hydroxyl group, this
model potentially explains the structure activity relationship
difference between PP1 and AB129 in terms of CBR1 inhibition. A
conclusion from these data is that AB129 is a potent inhibitor of
CBR1, a member of the SDR family of enzymes, which are responsible
for a number of important small molecule metabolic steps in a
variety of organs and cell types.
GSH modified prostaglandins were recently discovered in colorectal
cancer cells. (FIG. 31; Biochim Biophys Acta 1584: 3745, 2002) CBR1
has a glutathione binding site distinct from the AB129 binding
site. Glutathione binding activity of wild type and N90V mutant
CBR1 was tested. The results demonstrate that glutathione binding
activity is separate from the AB129 binding activity as
demonstrated for the N90V mutant CBR1. FIG. 32. These results
indicate that a mechanism exists for inhibition of CBR1 and
prostaglandin synthesis which can be effective in inhibition of
proliferation of colorectal cancer cells.
EXAMPLE 5
Does Inhibition of CBR1 with AB129, Increase the Cell Killing
Potency of Daunorubicin?
One important cellular function of CBR1 is to metabolize
xenobiotics such as the anticancer agent daunorubicin, a member of
the anthracyclin antibiotic agents including adriamycin.
Experiments were performed to test whether daunorubicin and AB129
treated cells were capable of exhibiting cell toxicity at
concentrations lower than that needed for each individual compound
to induce cell death alone. In agreement with this therapeutic
strategy, A549 cells were treated with 0.4 .mu.M of AB129 which led
to a 15% loss of viability after two days of treatment (FIGS.
33-34). Similarly, daunorubicin, as a single agent, when added to
A549 cells at 0.8 .mu.M let to a similar 15% loss of viability of
A549 cells. However, when the two drugs, AB129 and daunorubicin
were added in combination at 0.4 .mu.M and 0.8 .mu.M, respectively,
a decrease in almost 70% of A549 cell viability was observed (FIGS.
33-34). This experiment suggests that AB129 is capable of enhancing
the potency of daunorubicin mediated cancer cell killing. Moreover,
since AB129 does this through inhibition of CBR1, the toxic
metabolite of daunorubicin, daunorubicinol is not produced and thus
in vivo the cardiotoxic effects of daunorubicinol, or other
anthracyclin anti-cancer therapy should be enhanced.
EXAMPLE 6
Other Targets of AB129, Including Potential Protein Kinases.
AB129 is an analog of PP1, the Src family protein kinase inhibitor.
Experiments were performed to determine whether AB129 was capable
of inhibiting the Src family kinase, Fyn. In fact AB129 is a potent
(10 nM IC.sub.50) inhibitor of Fyn. While the ability of AB129 to
inhibit protein kinases as well as CBR1 could be important for its
biological activity in some settings, AB129 compound was modified
to produce a pure CBR1 inhibitor. Such a compound would serve as a
test compound for determining the importance of dual inhibition of
CBR1 and protein kinases. The crystal structure of PP1 bound to
Hck, a Src family kinase, shows that the exocyclic amine of PP1
makes a key H-bond interaction with a back-bone carbonyl group of
Hck in the ATP binding pocket. It was predicted that addition of a
methyl group to this amine of AB129 would disrupt this H-bond
interaction because it would point away from the phenyl ring, thus
eliminating the H-bond donor of AB129. In fact, the resulting
analog of AB129, RB6 (FIG. 35) was found to be equipotent as a CBR1
inhibitor, yet is predicted to be >100 fold less potent as an
inhibitor of Fyn. Anti-Fyn IC.sub.50 for RB6 was 70 .mu.M. Anti-Fyn
IC.sub.50 for AB129 was 10 nM The ability to generate inhibitors
for protein kinases (PP1), or CBR1 (RB6), or both targets (AB129)
(FIG. 35), should help in distinguishing between the cellular
effects of CBR1 and/or kinase inhibition.
EXAMPLE 7
Designing New Potent and Selective Inhibitors of SDR Family Members
Including Carbonyl Reductase 1 (CBR1), and 11.beta.-Hydroxysteroid
Dehydrogenase 1 and 2 (11.beta.-HSD1 and 2).
Incorporating SAR data obtained from a small set of synthesized
compounds, and allowing as yet untried diversity elements, a
library of putative CBR inhibitors was envisioned that conserves
the putative pharmacophore but introduces structural diversity
elements that might increase the affinity and specificity of the
library members toward various SDR enzymes. The proposed library
utilizes a pyrrolopyrimidine scaffold as opposed to the
pyrrazolopyrimidine scaffold of AB129, and the compounds
synthesized thus far indicate the anti-CBR activity of both of
these scaffold types are comparable. The docked AB129-CBR structure
was used to inform the choice of library substituents indicated in
FIG. 36.
Diversity element R.sup.3 or R.sup.4 (Formula I) include either
proton or alkyl substituents. Compounds with H-bond donors at this
position can inhibit kinases, as does AB129, so the presence of
alkyl substituents at this position should shift affinity away from
kinases. AB129 and the analogs synthesized thus far possess
saturated alkyl substituents at R.sup.2. In addition to such
substituents, the library will also include negatively charged
substituents or planar aromatic groups at this position. The
docking model indicates the t-butyl group (analogous to the
position of R.sup.2) of AB129 is solvent exposed, and in close
proximity to one lysine and two arginine residues forming the
binding cavity for the NADP(H) phosphate. To elicit potential
ion-pairing interactions, negatively charged groups like
p-cyclohexanoic acid will be included. Also adjacent to the t-butyl
group of AB129 is the NADP(H) adenine binding cavity. A substituent
at R.sup.2 favors an orientation allowing access to this cavity.
Thus, planar aromatic substituents such as indole could be
accommodated resulting in increased affinity. Diversity elements at
R.sup.1 will include substituted phenyl, indole and thiophene
moieties selected for potential H-bonding and charge interactions
with specific active site residues. Substituents larger than the
AB129 phenoxy may gain additional affinity by exploiting van der
Walls interactions deeper within the NADPH binding channel.
Although all H-bond interactions predicted between AB129 and CBR
are made to conserved residues, the binding orientation and
adjacent residue identity can vary throughout the SDR family. These
interactions can be of particular interest for tailoring the
specificity of these compounds to different enzymes of the SDR
class.
As mentioned above, it was postulated that both pyrazolopyrimidines
like AB129 and analogous pyrrolopyrimidines would have comparable
anti-CBR activity. In order to verify this assumption, the
pyrazolopyrimidine RB2 and the analogous pyrrolopyrimidine RB5 were
synthesized (FIG. 37). These compounds employ an R.sup.2 isopropyl
as opposed to the AB129 t-butyl because the Mitsunobu reaction used
for RB5 synthesis is not amenable to t-butyl alkylation. Both of
these compounds inhibit CBR with similar affinity (IC.sub.50 values
comparable to AB129) and kill adenocarcinoma cells in culture.
The library of pyrrolopyrimidines well suited for SDR inhibition is
constructed using the following solution and solid-phase reactions
(FIG. 38). The pyrrolopyrimidine scaffold
4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine, 5, was chosen for its
synthetic utility and literature precedent. This scaffold has been
synthesized previously (Pudlo, J. S., et al., J Med Chem, 33:
1984-92, 1990. Haslam, R., in U.K. Patent 812,366, 195 9: U.K), and
was synthesized in our laboratory from ethyl cyanoacetate and
bromoacetaldehyde diethyl acetal in six steps (10% yield). The
library synthesis will involve introduction of R.sup.2 by Mitsunobu
alkylation of the scaffold, 5, using solution-phase chemistry, and
a resin will be loaded with an R.sup.3 R.sup.4 primary amine by
reductive amination. Combining these materials and heating will
allow S.sub.NAr capture of the alkylated scaffold. Finally, a
solid-phase Suzuki coupling to introduce R.sup.3 and TFA mediated
cleavage should yield the library members. Similar reaction
conditions and the use of the scaffold, 5, are also amenable to
solution-phase syntheses.
Primary amines containing diversity element R.sup.1 will be coupled
to 4-formyl-3,5-dimethoxyphenoxymethyl-functionalized (PAL) resin
to yield 7 in a manner analogous to published conditions (Moon, H.
S., et al., Journal of the American Chemical Society, 124:
11608-11609, 2002). When a primary amine at R.sup.3or R.sup.4 is
required, a protecting strategy can be employed. Thus, acid-labile
2,4,6-trimethoxybenzylamine should be suitable for this use. This
amine mirrors the structure of the functionalized resin and should
be equally acid sensitive during the cleavage reaction. Separately,
in solution phase, diversity element R.sup.2 will be introduced by
Mitsunobu alkylation (Ding, S., et al., J Org Chem, 66: 8273-6,
2001) of 5 to produce 6. Although Mitsunobu alkylation has been
demonstrated on solid phase (Ding, S., et al., J Am Chem Soc, 124:
1594-6, 2002. Ding, S., et al., J Comb Chem, 4: 183-6, 2002), 6 was
prepared in solution. A similar coupling and reductive amination
strategy using a purine scaffold has been developed for the
synthesis of derivatized purines used as kinase inhibitors
(Ugarkar, B. G., et al., J Med Chem, 43: 2894-905, 2000). In
parallel, each of the resultant products 6 will be reacted with the
resin bound amine 7 to yield compound 8 on solid support. Again in
a manner analogous to that used for the preparation of kinase
inhibitors, 8 will be treated with commercially available boronic
acids using Suzuki coupling conditions to introduce the diversity
element R.sup.1. Similar reactions have been carried out in
solution phase (Ugarkar, B. G., et al., J Med Chem, 43: 2894-905,
2000). The compounds can then be cleaved from the PAL resin using
trifluoroacetic acid.
Optimizations of both reductive amination and scaffold loading
(FIG. 20) have been performed using a number of conditions, and
good results have been obtained. The reductive amination reactions
to produce 7 have utilized methylamine, ethylamine, benzylamine and
2,4,6-trimethoxybenzylamine (FIG. 39). The reactions were carried
out in peptide synthesis cartridges using the reducing agent
NaBH(OAc).sub.3 in a manner analogous to that previously reported
(Moon, H. S., et al., Journal of the American Chemical Society,
124: 11608-11609, 2002). When using 2,4,6-trimethoxybenzylamine the
HCl salt was used, and a stoichiometric quantity of DIEA was also
included. Comparable yields were obtained using 5 and 20 eq. (0.1
and 0.4 M respectively) of amine with either THF or 1:1 THF:DMF as
evidenced by FMOC quantitation (Bunin, B., The Combinatorial Index,
ed. San Diego: Academic Press, 1998).
Loading of the scaffold with resin bound primary amine 7 was also
attempted using each of the amine-loaded resins. Either THF or
n-BuOH at 60 or 90.degree. C. respectively in the presence of 10%
DIEA was employed. The values reported (FIG. 39) represent
conversion at 90.degree. C. for 18 h, as determined by FMOC
quantification.
The conditions for both reductive amination and resin loading
appear to work well for the conditions tested.
The pyrrolopyrimidine scaffold 5 was also used as starting material
for the solution phase synthesis of RB5 and RB6 (FIG. 10) with
greater than 50% overall yield. Thus, should library members
demonstrate activity in vitro and larger quantities of material are
needed, a solution-phase strategy can be more efficient. RB5
synthesis commenced with Mitsunobu alkylation of 5 with 2-propanol
resulted in the preparation of 6 in greater than 90% yield.
Mitsunobu reactions of pyrrolopyrimidines are scarce in the
literature; however, the utility of the transformation has been
demonstrated with purines. Therefore, an analogous procedure using
DiAD, PPh.sub.3 and 2-propanol was employed (Ding, S., et al., J
Org Chem, 66: 8273-6, 2001). Compound 6 was subsequently aminolyzed
at elevated temperature in a sealed vessel using a saturated
methanolic ammonia solution. As expected, this reaction was
selective for the 4-chloro position of 6 for both ammonia and
methylamine used during the synthesis of RB5 and RB6 respectively.
Treatment of the products with 3-(hydroxyphenyl)boronic acid
afforded RB5 and RB6 using available Suzuki coupling conditions
(Moon, H. S., et al., Journal of the American Chemical Society,
124: 11608-11609, 2002). These conditions do not require the
protection of hydroxylic or amino substituents of the boronic
acids.
In vitro assays for the measurement of CBR activity have been
developed (Bohren, K. M., et al., J Mol Biol, 244: 659-64, 1994)
and used in our laboratories. CBR was expressed in E. coli and
purified using glutathione beads; CBR has a naturally occurring.
glutathione binding site. An N-terminal 6-His tagged protein was
prepared to allow purification by metal affinity. Our CBR assay
employs the synthetic substrate Menadione
(2-methyl-1,4-naphthoquinone). Reaction progress is monitored by
the decrease in NADPH-absorbance at 340 nm. Saturating
concentrations of Menadione with variable concentrations of NADPH
are employed in order to ascertain K.sub.I values.
A high-throughput assay will be optimized for analyzing library
compounds. A 96-well format will be employed, and the disappearance
of NADPH will be monitored either by fluorescence or absorbance.
Using fixed substrate and enzyme concentrations, IC.sub.50 values
for library members can be obtained. Cell culture assays for CBR
inhibition can also be developed, as CBR inhibitors in the presence
of daunorubicin would be expected to lead to a further decrease in
cell proliferation rate than either compound alone.
In addition to the use of CBR activity for screening library
compounds, recombinant 11.beta.-HSD1 is prepared (Nobel, C. S., et
al., Protein Expr Purif, 26: 349-56, 2002). This enzyme is a
membrane-bound glycoprotein, and previous reports indicate that the
isolation of active enzyme is not trivial. An expression system
using Pichia pastoris has been described. A yeast expression vector
for 11.beta.-HSD1incorporating an N-terminal 6-His tag and a
picornavirus protease cleavage site. Following characterization of
the enzyme activity, an assay similar to that used for detecting
CBR activity is developed. An expression system for 17.beta.-HSD1
is developed to allow the specificity of these compounds to be
further investigated.
The inhibitor screening results should provide valuable SAR data
for the pyrrolopyrimidine pharmacophore, and provide valuable
information for producing even more effective inhibitors in the
future. Continuing studies to assess the selectivity of these
analogs among different SDR enzymes and associated cellular
phenotypes will be pursued. Differential activity of the library
members toward CBR and other SDR members when coupled with
available crystallographic and sequence data, should help to
identify important structural and electronic features that lead to
effective and specific inhibition of SDR enzymes.
Chemical genetic screens for small molecules which target disease
related biological processes hold great promise for development of
future medicines. A well designed chemical genetic screen like a
well designed genetic screen requires manipulation of the pathway
of interest such that early-low potency "hits" can be identified.
This precludes the simultaneous screening of more than one pathway
in each assay. Since a limited portion of chemical space is probed
in any library of small molecules, there is a limited chance that a
potent and selective agent targeting a pathway of interest will be
present. Consequently the frequency of identifying true drug-leads
in such screens has been relatively low. Chemical-genetic screens
have an additional challenge, compared with genetic screens, that
of target identification: Identification of the target of a small
molecule lead compound is difficult because the affinity of such
early hits are often low, and thus not amenable to successful
affinity purification strategies which require tight, or
irreversible inhibitors (usually only found in natural products or
advanced drug development candidates). To overcome these problems,
the traditional format of chemical genetic screens was inverted.
Rather than screening a very large collection of small molecules
for antagonists or agonists of a single pathway, a small panel of
compounds was screened for the ability to perturb any pathway in a
panel of cell lines with high potency and selectivity. A strategy
was exploited utilizing a cell morphology-microscopy based assay
coupled with an automated image analysis algorithm designed to
detect perturbations to a great many cell processes simultaneously
including, for example, cell cycle arrest point, cytoskeletal
structure, cell adhesion status, organelle organization. This
approach allowed phenotypic effects of all members of the library
to be scored, and showed that almost every compound in the library
at the highest doses analyzed (10 .mu.M), produced some phenotypic
effects. AB129 was selected, which potently produced a novel
phenotype, (several controls were included such as, Taxol and
K252a, to define known phenotype signatures), in a single cell
line-the lung cancer A549 line, but not other cell lines. Using
traditional target identification methods applied to natural
products, but rarely applied to first generation hits from chemical
genetic screens the target of AB129 in A549 lysates was identified
as an NADPH dependent reductase, carbonyl reductase 1 (CBR1). CBR1
serves a dual role of prostaglandin biosynthesis and xenobiotic
metabolism. In vitro assays demonstrated AB129 is an NADPH
competitive inhibitor of CBR1 with a Ki of approximately 300 to 400
nM, validating the overall approach to be successful at
identification of potent lead compounds. The relevance of CBR1 to
lung cancer was explored through analysis of transcriptional
profiling data of various lung cancer cell lines. This analysis
revealed CBR1 to be a highly upregulated transcript in
adenocarcinomas suggesting it might play an important role in
producing prostaglandins as autocrine factors for A549 cell
survival. siRNA studies confirm that CBR1 is essential for A549
cell viability, confirming the mode of action of AB129 at inducing
A549 cell death. To independently confirm the ability of AB129 to
inhibit CBR1 in A549 cells, an assay based on the role of CBR1 in
attenuating the anti-cancer action of daunorubicin was employed.
Indeed, AB129 is able to potentiate daunorubicin action in A549
cells, suggesting the former can be an attractive combination
therapy with daunorubicin. Moreover, AB129 is able to block
production of the cardiotoxic metabolite daunorubicinol from
daunorubicin. Thus, a new broad based phenotype profiling method
allowed for system wide screening of chemical libraries allowed for
the discovery of a potent small molecule capable of selective
killing of lung cancer A549 cells and potentiating the action of
daunorubicin.
EXAMPLE 8
New Potent and Selective Inhibitors of SDR Family Members Including
Carbonyl Reductase 1 (CBR1), and 11.beta.-Hydroxysteroid
Dehydrogenase 1 and 2 (11.beta.-HSD1 and 2).
##STR00020##
Compound RB8 employs a substituent, benzyl, at the exocyclic amine
on the pyrrolopyrinidine/pyrazolopyrimidine scaffold. The anti-CBR
IC.sub.50=4.4 .mu.M, and the anti-Fyn IC.sub.50=20 .mu.M.
##STR00021##
Compound RB11 employs a carboxy alkyl substituent at N-9 of the
pyrrolopyrinidine/pyrazolopyrimidine scaffold. Compound RB11
demonstrates an improved anti-CBR IC.sub.50 activity. An increased
affinity can be attributed to potential hydrogen bond interactions
between the carboxylate and charged residues including Asn 13, Arg
41, and Arg 37 of CBR. These residues would otherwise interact with
the NADPH 3'-OPO.sub.3.sup.2- phosphate upon substrate binding, and
can provide specificity for short chain dehydrogenase/reductase
(SDR) utilizing NADP(H) rather than NAD(H). The anti-CBR IC.sub.50
for compound RB11 is 220 nM.
##STR00022##
RB10 is an intermediate in the synthesis of RB11. The anti-CBR
IC.sub.50=1.15 .mu.M. An improved IC.sub.50 for compound RB10 may
be due to its inability to hydrogen bond to residues including Asn
13, Arg 41, and Arg 37 of CBR1.
Substituents at N-9 of the pyrrolopyrimidine/pyrazolopyrimidine
scaffold can further include alkyl chains substituted with carboxyl
and/or phosphoryl, e.g., R.sub.2 substituents of compounds of
Formulas I, II, or III.
In addition to the above compounds, the effect of substituents at
the meta position of the phenyl ring have been studied (see below,
Compound A, as an example of a compound derived from Formula I).
Potency as anti-CBR activity can be increased with electron
withdrawing groups (Br, CF.sub.3) at the 5-position of the
pyrrolopyrinidine/pyrazolopyrimidine scaffold. The CBR binding can
be tolerant of even large substituents (tert-butyl) at this
position.
Substituting a halo substituent, for example, chloro or bromo, in
place of the exocyclic amine (see below, Compound B. as an example
of a compound derived from Formula III) provides an increased
affinity for CBR binding. Compounds of the present invention can
employ an exocyclic amine or a halo substituent as part of the
pyrrolopyrinidine/pyrazolopyrimidine scaffold. Substituting a
chloro substituent for a methylamino substituent on the
pyrrolopyrinidine/pyrazolopyrimidine ring gives a roughly 10-fold
increase in potency. For substituents of chloro or bromo, the
IC.sub.50 is approximately 30 nM.
##STR00023##
EXAMPLE 8
New Potent and Selective Inhibitors of SDR Family Members Including
Carbonyl Reductase 1 (CBR1), and Src Family Protein Kinase,
Fyn.
A chloro substituent in place of the exocyclic amine on the
pyrrolopyrimidine/pyrazolopyrimidine scaffold provides increased
affinity. See SD1 and SD5 below. The switch from methylamino to
chloro substituents on the pyrimidine ring gives a roughly 10-fold
increase in potency in all cases. For SD1 compound, having
methylamino and bromo substituents, the anti-CBR IC.sub.50 is 220
nM. For SD5 compound, having chloro and bromo substituents, the
anti-CBR IC.sub.50 is 27 nM.
##STR00024##
Because replacement of the exocyclic amine with a more hydrophobic,
electron-withdrawing substituent (Cl) increases potency, these
results suggest that exocyclic methlyamino can be substituted with
halogens (F, Cl, Br, I), hydrogen, small electron-withdrawing
groups (NO.sub.2, CN, etc.), or small alkyl and haloalkyl groups at
this position.
In addition the effect of substituents at the meta position of the
phenyl ring increases potency as a CBR1 inhibitor (see below).
Potency as a CBR1 inhibitor increases with electron withdrawing
groups (Br, CF.sub.3) at the 5-position and the CBR seems tolerant
of even large substituents (t-butyl) at this position. substituents
at the meta position include electron withdrawing groups, for
example, ester and amide linkages (--COOR, --CONHR).
##STR00025##
The anti-CBR IC.sub.50 for SD2 is 3.04 .mu.M. The anti-CBR
IC.sub.50 for SD6 is 193 nM
##STR00026##
The anti-CBR IC.sub.50 for SD3 is 7.65 .mu.M. The anti-CBR
IC.sub.50 for SD7 is 376 nM.
##STR00027##
The anti-CBR IC.sub.50 for SD4 is 416 nM. The anti-CBR IC.sub.50
for SD8 is 67 nM.
EXAMPLE 9
New Potent and Selective Inhibitors of SDR Family Members Including
Carbonyl Reductase 1 (CBR1), and Src Family Protein Kinase,
Fyn.
Table 1 shows compounds of the present invention that are
inhibitors of the Src family knase, Fyn, and are inhibitors of
carbonyl reductase 1 (CBR1). AB129 is a potent (10 nM IC.sub.50)
inhibitor of Fyn. While the ability of AB129 to inhibit protein
kinases as well as CBR1 could be important for its biological
activity in some settings. AB129 compound was further modified to
produce compounds of the present invention which are CBR1
inhibitors with reduced inhibitory activity for Fyn.
TABLE-US-00001 TABLE 1 IC.sub.50 for anti cFYN and anti-hCBR1
Compound Structure IC.sub.50 anti-c-Fyn-wt IC.sub.50 anti-hCBR1
AB001 ##STR00028## 110 nM >20 .mu.M AB060 ##STR00029## 50 nM
>20 .mu.M AB061 ##STR00030## 50 nM >20 .mu.M AB129
##STR00031## 8 nM 790 nM PP1 ##STR00032## 50 nM >20 .mu.M MT13
##STR00033## 5 .mu.M >20 .mu.M MT15 ##STR00034## 0.2 .mu.M 930
nM MS01 ##STR00035## 3 nM 1 .mu.M RB01 ##STR00036## 1.2 .mu.M
>20 .mu.M RB02 ##STR00037## 11 nM 1 .mu.M RB03 ##STR00038## ~20
.mu.M >20 .mu.M RB04 ##STR00039## 120 nM >20 .mu.M RB05
##STR00040## 12 nM 760 nM RB06 ##STR00041## 70 .mu.M 590 nM RB07
##STR00042## not determined (n/d) >20 .mu.M RB08 ##STR00043## 20
.mu.M 4.4 .mu.M RB09 ##STR00044## n/d >20 .mu.M RB10
##STR00045## n/d 1.15 .mu.M RB11 ##STR00046## n/d 220 nM SD1
##STR00047## n/d 220 nM SD2 ##STR00048## n/d 3.04 .mu.M SD3
##STR00049## n/d 7.65 .mu.M SD4 ##STR00050## n/d 416 nM SD5
##STR00051## n/d 28 nM SD6 ##STR00052## n/d 193 nM SD7 ##STR00053##
n/d 376 nM SD8 ##STR00054## n/d 67 nM
When ranges are used herein for physical properties, such as
molecular weight, or chemical properties, such as chemical
formulae, all combinations and subcombinations of ranges and
specific embodiments therein are intended to be included.
The disclosures of each patent, patent application and publication
cited or described in this document are hereby incorporated herein
by reference, in their entirety.
Those skilled in the art will appreciate that numerous changes and
modifications can be made to the embodiments of the invention and
that such changes and modifications can be made without departing
from the spirit of the invention. It is, therefore, intended that
the appended claims cover all such equivalent variations as fall
within the true spirit and scope of the invention.
* * * * *